WO2004087765A1 - Titanium dioxide complex having molecule distinguishability - Google Patents

Titanium dioxide complex having molecule distinguishability Download PDF

Info

Publication number
WO2004087765A1
WO2004087765A1 PCT/JP2004/004638 JP2004004638W WO2004087765A1 WO 2004087765 A1 WO2004087765 A1 WO 2004087765A1 JP 2004004638 W JP2004004638 W JP 2004004638W WO 2004087765 A1 WO2004087765 A1 WO 2004087765A1
Authority
WO
WIPO (PCT)
Prior art keywords
titanium dioxide
molecule
ability
molecular
complex
Prior art date
Application number
PCT/JP2004/004638
Other languages
French (fr)
Japanese (ja)
Inventor
Shuji Sonezaki
Koki Kanehira
Shinichi Yagi
Yumi Ogami
Original Assignee
Toto Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toto Ltd. filed Critical Toto Ltd.
Priority to EP04724768A priority Critical patent/EP1614694A4/en
Priority to AU2004226053A priority patent/AU2004226053A1/en
Priority to JP2005504274A priority patent/JPWO2004087765A1/en
Priority to US10/551,164 priority patent/US20060281087A1/en
Publication of WO2004087765A1 publication Critical patent/WO2004087765A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Definitions

  • the present invention provides an endocrine disrupting substance, or a molecule having a specific binding ability to an etiological molecule or a cancer cell, and decomposing these substances, molecules, and cells by irradiation with ultraviolet light.
  • the present invention relates to a titanium dioxide composite having a discriminating ability.
  • anatase-type titanium dioxide has a photocatalytic action, and it is known that strong oxidizing power decomposes organic substances such as microorganisms, dirt, and odorous substances. In particular, it has a strong decomposing effect on hardly decomposable substances such as endocrine disrupting substances, and is expected to be effective for environmental purification (for example, Y. Ohko et al .: Environmen).
  • a device has been devised to increase the decomposition efficiency of titanium dioxide by combining titanium dioxide and an inorganic adsorbent such as activated carbon zeolites (see, for example, Japanese Patent Application Laid-Open No. H01- 1). No. 893222). Also in the surface treatment of titanium dioxide, promote the oxidation and reduction reactions of the photocatalyst by depositing a catalytic metal that promotes the reduction reaction such as palladium on the surface of the photocatalyst such as titanium dioxide. (See, for example, Japanese Patent Application Laid-Open No. 60-149440).
  • materials for selectively binding endocrine disrupting substances such as DNA have no means for reliably removing or decomposing bound endocrine disrupting substances, etc., and have a limited purification capacity due to the problem of adsorption saturation.
  • the above-mentioned device for enhancing the ability of titanium dioxide as a photocatalyst is not directed to binding or decomposition of a specific substance.
  • the target substance is identified and selectively bonded, and this is decomposed by the strong oxidizing power of the photocatalyst. The technology is not known.
  • DDS drug delivery system
  • Non-degradable polymers and amino acid polymers for example, JP-A-09-255590
  • ribosomes for example, JP-2003-226-63) No. 8
  • hollow nanoparticles of protein see, for example, Japanese Patent Application Laid-Open No. 2003-281986
  • Another example is a targeting DDS, a system that delivers the required amount of drug to the required site at the required time, and ultimately targets the lesion at a missile. Targeting drugs (missile therapy).
  • targeting is achieved by supporting ligands on DDS carriers and specifically recognizing and binding to receptors on the target cell surface.
  • the ligand corresponding to the receptor targeted for such active targeting include antigens, antibodies, peptides, glycolipids and glycoproteins.
  • the glycolipid-protein sugar chain is an information molecule in cell communication such as cell proliferation and differentiation, tissue regeneration and morphogenesis, biological defense and fertilization, or canceration and its migration. It has recently become clear that it plays an important role (see, for example, H Yamazaki et al .: Advanced Drug Derivery Review, 43, 225-244 (2000)).
  • titanium dioxide having a strong photocatalytic resolution to such a DDS
  • Yamazaki 6 Advanced Drug Der very Review, 43, 225-244 (2000), Japanese Patent Application Laid-Open No. 2000-200. 2—3169500, R. Cai et al .: Cancer Research, 52, 2346-2348 (1992)).
  • This, c titanium dioxide it is an after incorporated in bombarded metal particles such as gold carrying gun cells titanium dioxide targeting, try to kill cancer cells by irradiating light such as ultraviolet rays Is known to be extremely stable in air and in solution, and is safe and non-toxic in (shielded) animals.
  • the activation of titanium dioxide can be controlled by turning light on and off, so it is expected to be applied to DDS for cancer treatment.
  • titanium dioxide isoelectric point of titanium dioxide is around pH 6, and there is a problem that titanium dioxide particles aggregate under physiological conditions near neutrality. For this reason, it was not possible to administer titanium dioxide itself directly into blood vessels or use it as a carrier for DDS as it is. Further, there is no known technique for immobilizing a molecule having a selective binding ability such as the ligand on the surface of titanium dioxide, and it is currently difficult to apply titanium dioxide as DDS. In other words, in the field of medical science S, the “combination of molecular recognition ability and photocatalytic ability” The technique has not yet been developed due to the above problems. Related to the invention
  • the present inventors conducted intensive studies to solve the above-mentioned problems, and after modifying the surface of the titanium dioxide particles with a hydrophilic polymer, immobilized a molecule having a specific binding ability to the target molecule.
  • the present inventors have found that the converted titanium dioxide composite can achieve both molecular discriminating ability and photocatalytic ability, and completed the present invention.
  • the surface of the titanium dioxide fine particles is modified with a hydrophilic polymer, and the carboxyl group of the hydrophilic polymer and the titanium dioxide are bonded by an ester bond.
  • a molecule having specific binding ability to a target molecule is immobilized on a carboxyl residue of the hydrophilic polymer.
  • the resulting titanium dioxide complex having molecular discriminating ability has molecular discriminating ability for endocrine disrupting substances, pathogenic molecules, cancer cells, and the like, and exhibits a decomposition reaction of these substances by photocatalysis.
  • a titanium dioxide composite having the following formula: This complex has the ability to specifically identify and capture the target molecule in water or aqueous solution, and to strongly decompose it by ultraviolet irradiation or the like.
  • the properties of this complex such as its ability to be used in an aqueous solvent, being able to accurately identify and capture the target molecule, and exhibiting a strong photocatalytic activity, are those of harmful substances such as aqueous endocrine disruptors. It is extremely useful for degradation treatment or application to medical treatment IT 'such as destruction of specific etiological molecules or cancer cells.
  • FIG. 1 is a schematic diagram showing a titanium dioxide complex having a molecular discriminating ability according to the present invention.
  • FIG. 2 is a diagram showing the activity of degrading an antigen (-fete protein) by the anti-ffi-fete protein antibody-immobilized dihydrogenated protein complex of the present invention. Antigen degradation activity is indicated as a decrease in absorbance.
  • FIG. 3 is a diagram showing the results of evaluating the binding property between an anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention and an antigen (human serum albumin) by surface plasmon resonance.
  • an antigen human serum albumin
  • FIG. 4 is a graph showing the results of antigen (human serum albumin) degradation activity by the anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention.
  • the degrading activity of the antigen is expressed as the degradation rate (%) calculated from the decrease in the amount of the antigen bound to the antibody due to the degradation (measured by the surface plasmon resonance method).
  • a streptavidin-immobilized titanium dioxide complex was used as a control.
  • FIG. 1 is a schematic view showing a titanium dioxide composite having a molecular discriminating ability of the present invention. That is, the titanium dioxide composite having molecular discriminating ability of the present invention is obtained by dispersing titanium dioxide fine particles 1 and hydrophilic polymer 2 having a plurality of carboxyl groups in dimethylformamide to 90 to 180 °. After conducting a hydrothermal reaction for 1 to 12 hours with C to bond them with an ester bond, the molecule 3 having specific binding ability to the target molecule is fixed to the lipoxyl residue S of the hydrophilic polymer 2. It was made into.
  • the ester bond between the titanium oxide and the hydrophilic polymer is due to the fact that the titanium oxide on the particle surface is hydrated with water in the reaction system to form a hydroxyl group on the surface, and the hydroxyl group and the carboxyl group of the hydrophilic polymer are combined. React with each other to form an ester bond.
  • Ru can be applied various analytical methods, for example check the presence or absence of infrared absorption of 1 7 0 0 ⁇ ⁇ 8 0 near 0 cm 1 is an absorption band of infrared spectroscopy Nyo Riesuteru bond It is possible to do.
  • an amino group of the molecule 3 is mainly used.
  • the amino group can be introduced by an appropriate modification method, or a desired functional group or crosslink that is reactive with a carboxyl group other than the amino group can be introduced. It is also possible.
  • the titanium dioxide fine particles 1 used in the present invention have a dispersed particle size of 2 to 200 nm from the viewpoint of the degree of freedom of the form of use such as in the case of application to the inside of the body for the treatment of cancer or the treatment of cancer. Is desirable. Further, as the titanium dioxide used in the present invention, both anatase type and rutile type can be suitably used.
  • titanium dioxide having a desired crystalline system can be selected and used depending on the application, such as selecting an anatase type having strong photocatalytic activity.
  • titanium dioxide is present on at least a part of the particle surface, the characteristics of titanium dioxide on the particle surface are similar, even if the material is, for example, a composite material of a magnetic material and titanium dioxide. Therefore, it is possible to immobilize a molecule having a specific binding ability via a carboxyl group. Therefore, the magnetic material and titanium dioxide It is possible to produce a titanium dioxide composite having molecular discriminating ability by using the same method as in the case of a single titanium dioxide particle.
  • the hydrophilic polymer 2 used in the present invention is preferably a water-soluble polymer because it is assumed that the present titanium dioxide composite is used in a state of being dispersed in an aqueous solution.
  • any polymer having a plurality of carboxy groups can be used.
  • carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polystyrene, and the like can be used.
  • examples include carboxylic acids and copolymers (copolymers) having a carboxyl group unit.
  • polycarboxylic acids such as polyacrylic acid and polymaleic acid, and copolymers of acrylic acid / maleic acid and acrylic acid-sulfonic acid-based monomers are used.
  • a coalescence (copolymer) is more preferably used.
  • the titanium dioxide composite of the present invention can maintain a state of being uniformly dispersed in a wide range of PH regions including near neutrality due to the electric repulsion between the remaining carboxyl groups.
  • the molecule 3 having specific binding ability, which imparts molecular recognition ability to the titanium dioxide complex is not limited to the following as long as it is a molecule that specifically binds to the target molecule.
  • proteins are the most important molecules.
  • antibodies, ligands, receptor proteins, polysaccharide peptides, and even amino acids can be suitably immobilized as proteins.
  • titanium dioxide complex of simple protein For immobilization to the body, an amino group and a thiol group can be used, and in the case of a glycoprotein, an aldehyde group of a sugar can be used as a target functional group for immobilization.
  • bi-tin (or avidin) is introduced into the carboxyl group of titanium dioxide modified with a water-soluble polymer, and the protein is allowed to crosslink with avidin (or piotin). It is also possible to immobilize it using the interaction of bijin: avidin.
  • the titanium dioxide complex of the present invention can present a specific factor or ligand on the particle surface. Therefore, for example, it is possible to introduce the complex into a cell such as a cancer cell expressing a specific receptor by specifically binding the ligand: receptor.
  • factors and ligands include growth factors such as epidermal growth factor (EGF), transforming growth factor, platelet-derived growth factor, bone morphogenesis, nerve growth factor, etc., as well as interferon and interferon.
  • growth factors and ligands include growth factors such as epidermal growth factor (EGF), transforming growth factor, platelet-derived growth factor, bone morphogenesis, nerve growth factor, etc., as well as interferon and interferon.
  • hormones and ligands such as leukin, colony stimulating factor, tumor necrosis factor, erythropoietin, Fas antigen, and activin. These proteins can be immobilized as described above.
  • nucleic acid abmers that specifically bind to specific proteins.
  • Such an absormer can also be used as the molecule 3 having specific binding property that imparts the molecular discrimination ability of the present invention.
  • immobilizing nucleic acids the same procedure is performed by synthesizing modified DNA using an aminated primer, a biotinylated primer, and a thiolated primer during DNA amplification by polymerase chain reaction (PCR). It can be immobilized on the modified titanium dioxide by a method. For example, when aminated DNA is used for immobilization. An ester such as N-hydroxysuccinic acid imide (NHS) is introduced into the carboxyl group of the modified titanium dioxide in advance, and then re-aminated by nucleophilic substitution.
  • NHS N-hydroxysuccinic acid imide
  • DA can be covalently linked to the modified titanium dioxide.
  • the thiolated DNA is immobilized on the modified titanium dioxide by reacting the carboxyl group with NHS and then reacting with 2- (2-pyridinyldithiethane) ethaneamine. Is possible.
  • the carboxyl group of the molecule to be immobilized can be reacted with NHS, and then the molecule to be immobilized can be bound to the modified titanium dioxide by using hydrazine and reduced with sodium cyanoborohydride. .
  • the carboxyl group is converted into a bifunctional group by using a bifunctional hydrazide diamino-modified bitin, the avidin-modified molecule to be immobilized can be easily introduced onto the modified titanium dioxide.
  • the reagents, modification and cross-linking methods it is possible to easily immobilize a wide variety of molecules 3 having specific binding ability to the carboxyl residue introduced on the modified titanium dioxide. It is possible.
  • a functional group capable of binding to the carboxyl residue introduced on the modified titanium dioxide and the binding method is clear, a protein or a molecule having specific binding ability (3) can be used.
  • lipids and various physiologically active substances can be suitably used.
  • the titanium dioxide complex having molecular recognition ability of the present invention when it is to be applied to aqueous harmful substance treatment, medicine or medical treatment, it is uniformly dispersed in a neutral aqueous solvent due to physiological conditions in a living body. Is required.
  • the titanium dioxide composite having molecular recognition ability of the present invention since the titanium dioxide composite having molecular recognition ability of the present invention has a residual carboxy residue, the repulsive force due to the negative charge of the carboxyl group in the aqueous solvent is between the composites. Act on. Therefore, a wide pH range of pH 3 to 13 In the aqueous solution of the above, the composite can maintain a uniformly dispersed state without aggregation.
  • the titanium dioxide complex having molecular recognition ability of the present invention is dispersed in water, various pH buffer solutions, infusion solutions, or physiological saline. It becomes.
  • a softener or a spray containing the present dispersion can also be produced. This property is especially useful when applying titanium dioxide to DDS inside and outside the body. That is, since the dispersion of the titanium dioxide complex having molecular recognition ability of the present invention does not aggregate under physiological conditions near neutrality, it is injected into the vein directly injected into the affected tissue and targeted. Can be performed. In addition, it becomes possible to apply an ointment / spray containing the present dispersion liquid directly to an affected area such as the skin, and to perform light treatment using sunlight or an ultraviolet lamp.
  • the titanium dioxide complex having molecular recognition ability of the present invention can be used alone as DDS, and it can also be used as a form of DDS in other carriers.
  • the carrier in this case is not particularly limited, but ribosomes, virus particles, hollow nanoparticles and the like can be suitably used.
  • the light source device for exciting and activating the titanium dioxide composite having molecular discriminating ability of the present invention does not need to be special, but its wavelength is 400 nm or less due to the band gap of titanium dioxide. It is desirable. For external applications such as skin, sunlight, a normal ultraviolet lamp, or a black light can be suitably used.
  • the affected part of the body may be irradiated with ultraviolet rays by attaching an ultraviolet fiber to the endoscope.
  • the carbon dioxide having the molecular discriminating ability of the present invention as an action enhancer is considered. Titanium compound It is also possible to apply coalescence.
  • This dispersion was placed in a vial of 100 ml in volume and subjected to ultrasonic treatment at 200 Hz for 30 minutes.
  • the average particle size before and after the sonication was 36.4 nm and 20.2 nm, respectively.
  • the solution was concentrated to prepare a titanium dioxide sol (analysis type) having a solid component of 20%.
  • the obtained titanium dioxide sol is dispersed in 20 mI of dimethylformamide (DMF), and 0.2 g of polyacrylic acid (average molecular weight: 500, Wako Pure Chemical) is dissolved. After adding 1 O ml of the DMF, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel and hydrothermal synthesis was performed at 180 ° C for 6 hours. After the completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower. After taking out the solution, 80 ml of water was added and mixed with stirring.
  • DMF dimethylformamide
  • polyacrylic acid average molecular weight: 500, Wako Pure Chemical
  • Immobilization of anti-AFP antibody molecules on polyacrylic acid-modified titanium dioxide fine particles 1 ml of the polyacrylic acid-modified titanium dioxide sol (anatase type) obtained in Example 1 was exchanged using a desalting column PD10, and the solution was exchanged. 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water was obtained. In 1.5 ml of this solution, 200 1 ⁇ / 1 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinate imide (NHS ) was added and stirred for 10 minutes to activate carboxyl groups.
  • NHS N-hydroxysuccinate imide
  • Immobilization of anti-HSA antibody molecules on polyacrylic acid-modified titanium dioxide fine particles 1 ml of polyacrylic acid-modified titanium dioxide sol (anasease type) obtained in Example 1 was exchanged using a desalting column PD 10 Was carried out to obtain 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water.
  • a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinic imid (NHS) 0 1 ml was added and the mixture was stirred for 10 minutes to activate the carboxyl group.
  • Example 1 1 ml of the polyacrylic acid-modified titanium dioxide sol (anasease type) obtained in Example 1 was subjected to a solution exchange using a desalting column PD10, and 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water. Got. In 1.5 ml of this solution, a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) sorbodimide and 50 mM N-hydroxysuccinate imide (NHS). 1 ml was added and the mixture was stirred for 10 minutes to activate the carboxyl group.
  • N-hydroxysuccinate imide N-hydroxysuccinate imide
  • titanium tetraisopropoxide (Wako Pure Chemical Industries) was added to a final concentration of 0.05 M.
  • the reaction solution was centrifuged, and the precipitate was calcined at 350 ° C. for 2 hours. After baking, the mixture was dispersed in a 10 mM nitric acid aqueous solution, sonicated, and filtered with a 0.1 filter.
  • Magnetic material obtained 0.75 ml of composite titanium dioxide sol was dispersed in 20 ml of dimethylformamide (DMF), and polyacrylic acid (average molecular weight: 500, Wako Pure Chemical Industries) 0.3 After adding 1 ml of DMF in which g was dissolved, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel (HU-50, San-ai Science). Synthesis was performed at 180 ° C for 6 hours. After the completion of the reaction, the reaction vessel was cooled until the temperature of the reaction vessel became 50 ° C. or lower, and the solution was taken out into a separatory funnel. Next, 40 ml of black mouth was added, and the mixture was stirred and mixed to remove the lower layer, and the upper layer was recovered.
  • DMF dimethylformamide
  • polyacrylic acid average molecular weight: 500, Wako Pure Chemical Industries
  • This step was repeated twice to remove DMF.
  • 10 ml of the above solution 10 ml of 1.5 M NaCI, 20% (wZv) polyethylene glycol 600 (Wako Pure Chemical Industries) was added, and the supernatant was removed after centrifugation.
  • To the precipitate was added 2.5 ml of water, and the mixture was filtered through a Sephadex G_25 column to obtain a dispersion of polyacrylic continuously modified magnetic and composite titanium dioxide particles (anatase type).
  • anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG: MSU—304, Cosmo Bio Inc.) prepared in the same buffer was added to a concentration of 0.05 mg ZmI. After stirring at room temperature for 15 minutes, an aqueous solution of ethanolamine hydrochloride (pH 8.5) was added to a concentration of 0.5 M.
  • Example 2 0.75 ml of 20% titanium dioxide sol (anasease type) of the solid component obtained in Example 1 was dispersed in 20 ml of dimethylformamide (DMF), and acrylic acid / sulfone was added. Acid-based monomer copolymer (average molecular weight: 500, sample freeze-dried after substitution with toluene, Nippon Shokubai) 0.3 g of DMF dissolved in 10 mI was added, followed by stirring and mixing. . The solution was transferred to a hydrothermal reaction vessel (HU-50, San-ai Science) and reacted at 150 ° C for 5 hours.
  • DMF dimethylformamide
  • the reaction vessel was cooled to room temperature, and twice the amount of isopropanol (Wako Pure Chemical Industries) was added to the reaction solution. After leaving still at room temperature for 30 minutes or more, centrifugation was performed at 4000 X g for 20 minutes. The precipitate was collected. After washing this precipitate with 70% ethanol, 2.5 ml of water was added thereto to obtain an acrylic acid / nosulfonate-based copolymer-modified titanium dioxide sol (analysis type).
  • Acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol prepared in Example 7. 0.5 OmM / 20 OmM 1-ethyl-13- (3-dimethylaminopropyl) carbodiimide Then, 0.1 ml of a mixed solution of N-hydroxysuccinate imide (NHS) and 5 OmM of N-hydroxysuccinic acid was added, and the mixture was stirred for 10 minutes to activate the carboxyl group. After stirring, the mixture was equilibrated with 1 O mM acetate buffer (pH 5.0).
  • the solution was exchanged using PD 10 and 3 ml of a carboxyl group-activated acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) was added. Obtained.
  • an anti-DR4 monoclonal antibody Anti-TRAILR eceptor 1, mouse, code: SA-225, Funakoshi
  • an ethanolamine salt / salt aqueous solution pH 8.5
  • ⁇ -Fet protein (AFP, Cosmo Bio) was diluted with 50 mM PBS buffer (pH 7.0, Nippon Gene) to 1 Atg ZmI and prepared in Example 2.
  • An anti-AFP antibody-immobilized titanium dioxide complex was added so that the solid content was 0.101%. Then, the mixture was allowed to stand at 37 ° C. for 3 hours to form an aggregate by an antigen-antibody reaction. The formation of the aggregate of the AFP and the titanium dioxide complex immobilized with the anti-AFP antibody clearly indicates that the anti-AFP antibody immobilized titanium dioxide complex specifically recognizes and binds to AFP.
  • the ultraviolet light having a wavelength of 3 4 0 nm while stirring the aggregate is irradiated so that the 1 m W / cm 2, the absorption wavelength (Ido aggregate) in 6 0 0 nm measured by a spectrophotometer Specified.
  • the result is shown in figure 2.
  • Low aggregate concentration only during ultraviolet (UV) irradiation A decrease in absorbance is observed below. That is, it became clear that the antigen AFP was degraded by the photocatalysis of the titanium dioxide complex immobilized with the anti-AFP antibody.
  • This sensor chip was attached to a surface plasmon resonance measurement device: BIACORE 100 (BIACORE), and the HSA solution was passed at a flow rate of 10 nI / min, and then activated with 0.1 M ethanolamine. HSA was immobilized to produce an HSA-immobilized sensor chip.
  • BIACORE 100 BIACORE 100
  • HSA-immobilized sensor chip 0.1% of anti-HSA antibody-immobilized titanium dioxide complex sol prepared in Example 3 and 0.01% of streptavidin prepared in Example 4
  • the immobilized titanium dioxide complex sol was sent to confirm the antigen-antibody reaction. The results are shown in Figure 3.
  • Degradation of antigen HSA by anti-HSA antibody immobilized titanium dioxide complex HSA was diluted to a concentration of 20 ng / ml with a PBS buffer (pH 7.0, Nippon Gene), and the anti-HSA antibody-immobilized titanium dioxide complex prepared in Example 3 was added to a solid component of 0.1%. It was added so as to be 0 1%.
  • ultraviolet light having a wavelength of 340 nm was irradiated so as to have a wavelength of 1 mW / cm 2 , and sampling was performed every 15 minutes for 90 minutes. The same treatment was performed on the streptavidin-immobilized titanium dioxide composite prepared in Example 4.
  • an anti-HSA polyclonal antibody (Egret) immobilized sensor chip for surface plasmon resonance measurement was prepared.
  • each time-lapsed sample was sent to the anti-HSA antibody-immobilized sensor chip at 20 ⁇ .
  • the anti-HSA polyclonal antibody (Egret) was used as a secondary antibody.
  • 50 / zg / mI was sent at 1 I, sandwich was performed, and the RU value (corresponding to the amount of binding) 10 seconds after the antibody was sent was measured.
  • Figure 4 shows the decomposition rate of HS II calculated from the relative value when the RU value without UV irradiation was 100%. The results shown in FIG. 4 indicate that the anti-HSA antibody-immobilized titanium dioxide complex has a much higher HSA degradation rate than the streptavidin-immobilized titanium dioxide complex.
  • a titanium dioxide complex having a molecular discriminating ability, which specifically binds to endocrine disrupting substances, pathogenic molecules, cancer cells, and the like, and exhibits a decomposing action thereof by photocatalysis.

Abstract

A titanium dioxide complex capable of distinguishing molecules is obtained by modifying the surface of a fine titanium dioxide particle with a hydrophilic polymer in such a manner that titanium dioxide is bonded via an ester bond to a carboxyl group of the hydrophilic polymer and immobilizing a molecule having an ability to specifically bind to a target molecule to the carboxyl residue of the hydrophilic polymer. Due to the molecule distinguishability, this titanium dioxide complex can bind specifically to an endocrine disrupting chemical, a pathogenic factor, a cancer cell, etc. and decompose the same by a photocatalytic function.

Description

明細書 分子識別能を有する二酸化チタン複合你 技術分野  Description Titanium dioxide composite with molecular discrimination ability
本発明は、 内分泌搅乱物質、 あるいは病因分子やガン細胞などに対して特 異的な結合能を有する分子を固定化し、紫外線の照射などによってこれらの 物質、 分子、 細胞の分解作用を示す、 分子識別能を有する二酸化チタン複合 体に関する。 背景技術  The present invention provides an endocrine disrupting substance, or a molecule having a specific binding ability to an etiological molecule or a cancer cell, and decomposing these substances, molecules, and cells by irradiation with ultraviolet light. The present invention relates to a titanium dioxide composite having a discriminating ability. Background art
近年、 内分泌撹乱物質の分子識別能を有する D N Aなどの生体分子を支持 体上に固定化し、それにより選択的結合性を付与した材料が環境浄化材とし て提案されている （例えば、 特開 2 0 0 1 — 8 1 0 9 8号公報参照）。 また、 アナターゼ型ニ酸化チタンには光触媒作用があり、その強い酸化力により微 生物、 汚れ、 悪臭物質等の有機物を分解することが知られている。 特に、 内 分泌撹乱物質のような難分解性の物質に対しても強力な分解作用を示すこ とから、 環境浄化に有効であると期待されている （例えば、 Y. Ohko ら ： En v i ronmen t a l S c i en ce an d Tec hno l ogy, 35, 2365-2368 (2001 )参照）。 さ らに、現在では二酸化チタンと活性炭ゃゼ才ライ 卜などの無機吸着剤を複合 化することにより、二酸化チタンの分解効率を高めるような工夫がなされて いる （例えぱ特開平 0 1 — Ί 8 9 3 2 2号公報参照）。 二酸化チタンの表面 処理においても、パラジウムなどの還元反応促違触媒金属を二酸化チタン等 の光触媒表面に析出させることで、 光触媒の酸化、 還元反応を促進すること が考案されている （例えば、 特開昭 6 0 — 1 4 9 4 0号公報参照）。 In recent years, materials in which biomolecules such as DNA having the ability to identify molecules of endocrine disrupting substances are immobilized on a support, thereby imparting selective binding properties, have been proposed as environmental purification materials (see, for example, 0 0 1 — See 81 0 98 publication). In addition, anatase-type titanium dioxide has a photocatalytic action, and it is known that strong oxidizing power decomposes organic substances such as microorganisms, dirt, and odorous substances. In particular, it has a strong decomposing effect on hardly decomposable substances such as endocrine disrupting substances, and is expected to be effective for environmental purification (for example, Y. Ohko et al .: Environmen). tal Sci en ce and d Tec hno l ogy, 35, 2365-2368 (2001)). In addition, at present, a device has been devised to increase the decomposition efficiency of titanium dioxide by combining titanium dioxide and an inorganic adsorbent such as activated carbon zeolites (see, for example, Japanese Patent Application Laid-Open No. H01- 1). No. 893222). Also in the surface treatment of titanium dioxide, promote the oxidation and reduction reactions of the photocatalyst by depositing a catalytic metal that promotes the reduction reaction such as palladium on the surface of the photocatalyst such as titanium dioxide. (See, for example, Japanese Patent Application Laid-Open No. 60-149440).
しかしながら、 D N A等による内分泌攪乱物質の選択的結合材料について は、 結合させた内分泌攪乱钩質等の確実な除去や分解手段が無く、 かつ吸着 飽和の問題から浄化能力にも限界がある。 また、 前記の二酸化チタンの光触 媒としての能力を高めようとする考案についても、特定物質の結合や分解を 指向していない。 したがって、 例えば内分泌攪乱物質のみと選択的に結合し 分解することは不可能であった。 このように環境浄化の分野では、 目的の物 質のみを識別して選択的に結合し、 これを光触媒の強い酸化力によって分解 する、 すなわち二酸化チタンによる 「分子識別能と光触媒能との組合せ」 技 術は知られていない。  However, materials for selectively binding endocrine disrupting substances such as DNA have no means for reliably removing or decomposing bound endocrine disrupting substances, etc., and have a limited purification capacity due to the problem of adsorption saturation. Also, the above-mentioned device for enhancing the ability of titanium dioxide as a photocatalyst is not directed to binding or decomposition of a specific substance. Thus, for example, it was not possible to selectively bind and degrade only endocrine disrupters. As described above, in the field of environmental purification, only the target substance is identified and selectively bonded, and this is decomposed by the strong oxidizing power of the photocatalyst. The technology is not known.
一方、 近年医療分野における新しい薬剤投与形態として、 体内または体表 面で薬剤が経時的に徐放されるように設計されたシステム（ドラッグデリバ リーシステム ： D D S ) が注目されている。 これは、 既存医薬品の薬効を最 大限に高めると共に、その副作用を最小限に制御しょうとするものである。  On the other hand, in recent years, as a new drug administration form in the medical field, a system (drug delivery system: DDS) designed to release a drug over time in the body or body surface has attracted attention. This seeks to maximize the efficacy of existing drugs and to minimize their side effects.
D D Sにおける薬剤の担体としては、 非分解性高分子やアミノ酸ポリマー (例えば、 特開平 0 9 — 2 5 5 5 9 0号公報）、 リボソーム （例えば、 特開 2 0 0 3 — 2 2 6 6 3 8号公報参照）、 および夕ンパク質中空ナノ粒子 （例 えば、 特開 2 0 0 3 — 2 8 6 1 9 8号公報参照） 等が盛んに研究されている ( D D Sの概念をさらに進展させたものに、 標的指向 （ターゲティ ング） D D Sがある。 これは薬剤を必要な部位に、 必要な量を、 必要な時間に送り込む システ厶であリ、 最終的には病巣を確実に狙い撃ちするミサイルドラッグ (ミサイル療法） を目標としている。 Non-degradable polymers and amino acid polymers (for example, JP-A-09-255590) and ribosomes (for example, JP-2003-226-63) No. 8), and hollow nanoparticles of protein (see, for example, Japanese Patent Application Laid-Open No. 2003-281986) are being actively studied ( the concept of DDS is further advanced). Another example is a targeting DDS, a system that delivers the required amount of drug to the required site at the required time, and ultimately targets the lesion at a missile. Targeting drugs (missile therapy).
ミサイルドラッグの場合、 D D S担体にリガンドを担持させ、 標的細胞表 面に存在する受容体に特異的に認識 '結合させることによリタ一ゲティング を行う。 このような能動的ターゲティングの標的となる受容体に対応するリ ガンドとしては、 抗原、 抗体、 ペプチド、 糖脂質や糖タンパク質などが挙げ られる。 これらの中で、 糖脂質ゃ耱タンパク質の糖鎖は細胞の増殖や分化、 組織の ¾生や形態形成、 生体防御や受精機 、 あるいはガン化とその ¾移等 の細胞 ϋコミュニケーションにおける情報分子として重要な役割を果たし ていることが近年明らかになりつつある （例えば、 H Yamazaki ら： Advanced Drug Deri very Review, 43， 225-244 (2000)参照）。 In the case of missile drugs, targeting is achieved by supporting ligands on DDS carriers and specifically recognizing and binding to receptors on the target cell surface. I do. Examples of the ligand corresponding to the receptor targeted for such active targeting include antigens, antibodies, peptides, glycolipids and glycoproteins. Among these, the glycolipid-protein sugar chain is an information molecule in cell communication such as cell proliferation and differentiation, tissue regeneration and morphogenesis, biological defense and fertilization, or canceration and its migration. It has recently become clear that it plays an important role (see, for example, H Yamazaki et al .: Advanced Drug Derivery Review, 43, 225-244 (2000)).
このような D D Sに、強い光触媒分解能を有する二酸化チタンを応用しよ うとする試みがなされている ( . Yamazaki 6 ： Advanced Drug Der i very Review, 43, 225-244 (2000)、 特開 2 0 0 2— 3 1 6 9 5 0号公報、 R. Cai ら ： Cancer Research, 52, 2346-2348 (1992)参照）。 これは、 標的とするガ ン細胞に二酸化チタンを担持した金などの金属粒子を撃ち込んで取り込ま せた後、紫外線等の光を照射してガン細胞を死滅させようとするものである c 二酸化チタンは、 大気中や溶液中でも極めて安定な物質であり、 かつ （遮光 された） 動物体内では毒性もなく安全なことが知られている。 しかも、 二酸 化チタンの活性化を光のオン ·オフで制御することが可能なため、 ガン治療 に向けての D D Sへの応用が期待される。 Attempts have been made to apply titanium dioxide having a strong photocatalytic resolution to such a DDS (Yamazaki 6: Advanced Drug Der very Review, 43, 225-244 (2000), Japanese Patent Application Laid-Open No. 2000-200). 2—3169500, R. Cai et al .: Cancer Research, 52, 2346-2348 (1992)). This, c titanium dioxide it is an after incorporated in bombarded metal particles such as gold carrying gun cells titanium dioxide targeting, try to kill cancer cells by irradiating light such as ultraviolet rays Is known to be extremely stable in air and in solution, and is safe and non-toxic in (shielded) animals. In addition, the activation of titanium dioxide can be controlled by turning light on and off, so it is expected to be applied to DDS for cancer treatment.
しかしながら、 二酸化チタンの等電点は p H 6前後であり、 中性付近の生 理的条件下では二酸化チタン粒子が凝集してしまう問題点がある。 このため, 二酸化チタン自体を直接血管内に投与したり、そのままで D D Sの担体とし て用いることは不可能であった。 また二酸化チタン表面に、 前記リガンド等 の選択的結合能を有する分子を固定化する技術も知られておらず、二酸化チ タンの D D Sとしての宾用化は現状では囫難な状況にある。 すなわち、 医 S 分野においても二酸化チタンによる 「分子識別能と光触媒能との組合せ」 技 術は、 上記問題点のために未だ開発されていない。 発明の關示 However, the isoelectric point of titanium dioxide is around pH 6, and there is a problem that titanium dioxide particles aggregate under physiological conditions near neutrality. For this reason, it was not possible to administer titanium dioxide itself directly into blood vessels or use it as a carrier for DDS as it is. Further, there is no known technique for immobilizing a molecule having a selective binding ability such as the ligand on the surface of titanium dioxide, and it is currently difficult to apply titanium dioxide as DDS. In other words, in the field of medical science S, the “combination of molecular recognition ability and photocatalytic ability” The technique has not yet been developed due to the above problems. Related to the invention
本 ¾明者らは上記課題を解決するために銳意検討を行い、二酸化チタン徽 粒子表面を親水性高分子で修飾した後、 さらに目的分子に対して特異的な結 合能を有する分子を固定化した二酸化チタン複合体が、分子識別能と光触媒 能を両立できることを見い出し、 本発明を完成した。  The present inventors conducted intensive studies to solve the above-mentioned problems, and after modifying the surface of the titanium dioxide particles with a hydrophilic polymer, immobilized a molecule having a specific binding ability to the target molecule. The present inventors have found that the converted titanium dioxide composite can achieve both molecular discriminating ability and photocatalytic ability, and completed the present invention.
すなわち、 本発明の分子識別能を有する二酸化チタン複合体は、 二酸化チ 夕ン微粒子表面が親水性高分子により修飾され、該親水性高分子のカルボキ シル基と二酸化チタンはエステル結合で結合しているとともに、前記親水性 高分子のカルボキシル残基に目的分子に対して特異的な結合能を有する分 子を固定化したものである。 この方法により、 光触媒作用を有する二酸化チ タン粒子に、抗体などの特異的結合能を有する分子を導入することが可能と なリ、 分子識別能を有する二酸化チタン複合体を製造することができる。 結果として得られた分子識別能を有する二酸化チタン複合体は、内分泌撹 乱物質、 病因分子、 ガン細胞等に対する分子識別能を有し、 かつ光触媒作用 によりこれら物質の分解反応を示す、分子識別能を有する二酸化チタン複合 体を提供する。 本複合体は、 水または水溶液中で目的とする分子を特異的に 識別捕捉し、 紫外線照射などによりこれを強力に分解する能力を有する。 特 に、 本複合体が有する水系溶媒中で使用できる、 目的分子を正確に識別捕捉 できる、 かつ強力な光触媒能を示す等の特性は、 例えば水系の内分泌攪乱物 質を始めとする有害物質の分解処理、あるいは特定の病因分子やガン細胞の 破壊などの匿療分 IT'への応用に極めて有用である。 図面の簡単な説明 That is, in the titanium dioxide composite having molecular recognition ability of the present invention, the surface of the titanium dioxide fine particles is modified with a hydrophilic polymer, and the carboxyl group of the hydrophilic polymer and the titanium dioxide are bonded by an ester bond. In addition, a molecule having specific binding ability to a target molecule is immobilized on a carboxyl residue of the hydrophilic polymer. By this method, it is possible to introduce a molecule having a specific binding ability such as an antibody into titanium dioxide particles having a photocatalytic action, and it is possible to produce a titanium dioxide complex having a molecular discriminating ability. The resulting titanium dioxide complex having molecular discriminating ability has molecular discriminating ability for endocrine disrupting substances, pathogenic molecules, cancer cells, and the like, and exhibits a decomposition reaction of these substances by photocatalysis. A titanium dioxide composite having the following formula: This complex has the ability to specifically identify and capture the target molecule in water or aqueous solution, and to strongly decompose it by ultraviolet irradiation or the like. In particular, the properties of this complex, such as its ability to be used in an aqueous solvent, being able to accurately identify and capture the target molecule, and exhibiting a strong photocatalytic activity, are those of harmful substances such as aqueous endocrine disruptors. It is extremely useful for degradation treatment or application to medical treatment IT 'such as destruction of specific etiological molecules or cancer cells. BRIEF DESCRIPTION OF THE FIGURES
図 1 は、本発明の分子識別能を有する二酸化チタン複合体を示す模式図で あ 。  FIG. 1 is a schematic diagram showing a titanium dioxide complex having a molecular discriminating ability according to the present invention.
図 2は、 本¾明の抗 ffi -フエ 卜プロテイン抗体固定化二醸化チ夕ン複合体 による、 抗原 （ -フエ 卜プロテイン） の分解活性を示す図である。 抗原の 分解活性は、 吸光度の減少として表示されている。  FIG. 2 is a diagram showing the activity of degrading an antigen (-fete protein) by the anti-ffi-fete protein antibody-immobilized dihydrogenated protein complex of the present invention. Antigen degradation activity is indicated as a decrease in absorbance.
図 3は、本発明の抗ヒ 卜血清アルプミン抗体固定化二酸化チタン複合体と 抗原 （ヒ 卜血清アルブミン） との結合性を、 表面プラズモン共鳴法で評価し た結果を示す図である。 対照として、 ストレブ卜アビジン固定化二酸化チタ ン複合体を用いた。  FIG. 3 is a diagram showing the results of evaluating the binding property between an anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention and an antigen (human serum albumin) by surface plasmon resonance. As a control, a streptavidin-immobilized titanium dioxide complex was used.
図 4は、本発明の抗ヒ 卜血清アルブミン抗体固定化二酸化チタン複合体に よる、 抗原 （ヒ 卜血清アルブミン） の分解活性の結果を示す図である。 抗原 の分解活性は、 分解に伴う抗原の抗体との結合量の低下 （表面プラズモン共 鳴法で測定） から算出した分解率 （％) で表示されている。 対照として、 ス トレプ卜アビジン固定化二酸化チタン複合体を用いた。 発明を実施するための最良の形態  FIG. 4 is a graph showing the results of antigen (human serum albumin) degradation activity by the anti-human serum albumin antibody-immobilized titanium dioxide complex of the present invention. The degrading activity of the antigen is expressed as the degradation rate (%) calculated from the decrease in the amount of the antigen bound to the antibody due to the degradation (measured by the surface plasmon resonance method). As a control, a streptavidin-immobilized titanium dioxide complex was used. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の実施の形態を図面に基づいて具体的に説明する。図 1 は本発明の 分子識別能を有する二酸化チタン複合体を示す模式図である。 すなわち、 本 発明の分子識別能を有する二酸化チタン複合体は、二酸化チタン微粒子 1 と, カルボキシル基を複数有する親水性高分子 2をジメチルホル厶ァミ ドに分 散させて 9 0 ~ 1 8 0 °Cで〗 〜 1 2時間水熱反応を行って両者をエステル 結合で結合させた後、親水性高分子 2の力ルポキシル残 Sに目的分子に対し て特異的な結合能を有する分子 3を固定化させたものである。 ここで、 二酸 化チタンと親水性高分子とがエステル結合するのは、粒子表面の酸化チタン が反応系中の水に水和されてその表面に水酸基が生成し、その水酸基と親水 性高分子のカルボキシル基とが反応してエステル結合を形成することによ るものである。エステル結合の確認方法としては種々の分析方法が適用でき るが、例えば赤外分光法にょリエステル結合の吸収帯である 1 7 0 0〜 Ί 8 0 0 c m 1付近の赤外吸収の有無で確認することが可能である。 また、 特 異的な結合能を有する分子 3の固定化には、主にこの分子 3が有するァミノ 基が用いられる。 また、 ァミノ基の無い分子であっても適切な修飾方法によ リアミノ基を導入することは可能であり、あるいはアミノ基以外のカルボキ シル基と反応性のある所望の官能基や架橋を導入することも可能である。 本発明で用いる二酸化チタン微粒子 1 としては、凝集性の問題や癌治療用 として体内への適用の場合など、その使用形態の自由度の観点から分散粒経 が 2 ~ 2 0 0 n mであることが望ましい。 さらに、 本発明で用いる二酸化チ タンとしては、アナターゼ型およびルチル型のいずれも好適に使用可能であ る。 これは、 結晶系の異なる二酸化チタンであってもその化学的性質はほぼ 同一であり、 いずれの結晶系であっても水溶性高分子による修飾、 および特 異的な結合能を有する分子を固定化することが可能であるためである。例え ば、ガン細胞を破壊するためには光触媒活性の強いアナターゼ型を選択する など、用途に応じて所望の結晶系の二酸化チタンを選択し使用することがで きる。 An embodiment of the present invention will be specifically described with reference to the drawings. FIG. 1 is a schematic view showing a titanium dioxide composite having a molecular discriminating ability of the present invention. That is, the titanium dioxide composite having molecular discriminating ability of the present invention is obtained by dispersing titanium dioxide fine particles 1 and hydrophilic polymer 2 having a plurality of carboxyl groups in dimethylformamide to 90 to 180 °. After conducting a hydrothermal reaction for 1 to 12 hours with C to bond them with an ester bond, the molecule 3 having specific binding ability to the target molecule is fixed to the lipoxyl residue S of the hydrophilic polymer 2. It was made into. Where diacid The ester bond between the titanium oxide and the hydrophilic polymer is due to the fact that the titanium oxide on the particle surface is hydrated with water in the reaction system to form a hydroxyl group on the surface, and the hydroxyl group and the carboxyl group of the hydrophilic polymer are combined. React with each other to form an ester bond. Although as a confirmation method of ester bonds Ru can be applied various analytical methods, for example check the presence or absence of infrared absorption of 1 7 0 0~ Ί 8 0 near 0 cm 1 is an absorption band of infrared spectroscopy Nyo Riesuteru bond It is possible to do. For immobilization of the molecule 3 having a specific binding ability, an amino group of the molecule 3 is mainly used. In addition, even if the molecule does not have an amino group, the amino group can be introduced by an appropriate modification method, or a desired functional group or crosslink that is reactive with a carboxyl group other than the amino group can be introduced. It is also possible. The titanium dioxide fine particles 1 used in the present invention have a dispersed particle size of 2 to 200 nm from the viewpoint of the degree of freedom of the form of use such as in the case of application to the inside of the body for the treatment of cancer or the treatment of cancer. Is desirable. Further, as the titanium dioxide used in the present invention, both anatase type and rutile type can be suitably used. This is because the chemical properties of titanium dioxide with different crystal systems are almost the same, and the modification with a water-soluble polymer and the immobilization of a molecule with a specific binding ability are fixed in both crystal systems. This is because it is possible to make it possible. For example, in order to destroy cancer cells, titanium dioxide having a desired crystalline system can be selected and used depending on the application, such as selecting an anatase type having strong photocatalytic activity.
さらに、 二酸化チタンが少なくとも粒子表面の一部に存在すれば、 例えば 磁性材とニ酸化チタンとの複合材のようなものであっても、粒子表面の二酸 化チタンの特性は近似しているため、カルボキシル基を介した特異的結合能 を有する分子の固定化は可能である。 したがって、 磁性材とニ酸化チタンと の複合材であつても単一の二酸化チタン粒子の場合と全く同様の方法によ り、 分子識別能を有する二酸化チタン複合体を製造することが可能である。 本発明で用いる親水性高分子 2としては、本二酸化チタン複合体を水溶液 中に分散した状態で使用することを想定しているため、水溶性高分子である ことが望ましい。 本発明で用いる水溶性高分子としては、 複数のカルボキシ ル基を有する高分子であればいずれも適用可能であるが、例えばカルボキシ メチルデンプン、 カルボキシメチルデキス卜ラン、 カルボキシメチルセル口 ース、 ポリカルボン酸類、 およびカルボキシル基単位を有する共重合体 （コ ポリマー） などが挙げられる。 具体的には、 水溶性高分子の加水分解性およ び溶解度の観点から、 ポリアクリル酸、 ポリマレイン酸等のポリカルボン酸 類、およびアクリル酸/マレイン酸やアクリル酸 スルフォン酸系モノマー の共重合体 （コポリマー） がより好適に使用される。 これらの親水性高分子 によリニ酸化チタンを修飾することにより、親水性高分子のカルボキシル残 基に所望の特異的な結合能を有する分子 3を固定化することが可能となる。 さらに、分子 3の固定化後においても残余のカルボキシル基間の電気的斥力 により、本発明の二酸化チタン複合体は中性付近を含む広範囲の P H領域で 均一に分散した状態を維持できる。 Furthermore, if titanium dioxide is present on at least a part of the particle surface, the characteristics of titanium dioxide on the particle surface are similar, even if the material is, for example, a composite material of a magnetic material and titanium dioxide. Therefore, it is possible to immobilize a molecule having a specific binding ability via a carboxyl group. Therefore, the magnetic material and titanium dioxide It is possible to produce a titanium dioxide composite having molecular discriminating ability by using the same method as in the case of a single titanium dioxide particle. The hydrophilic polymer 2 used in the present invention is preferably a water-soluble polymer because it is assumed that the present titanium dioxide composite is used in a state of being dispersed in an aqueous solution. As the water-soluble polymer used in the present invention, any polymer having a plurality of carboxy groups can be used. For example, carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose, polystyrene, and the like can be used. Examples include carboxylic acids and copolymers (copolymers) having a carboxyl group unit. Specifically, from the viewpoint of hydrolysis and solubility of the water-soluble polymer, polycarboxylic acids such as polyacrylic acid and polymaleic acid, and copolymers of acrylic acid / maleic acid and acrylic acid-sulfonic acid-based monomers are used. A coalescence (copolymer) is more preferably used. By modifying the titanium dioxide with these hydrophilic polymers, it becomes possible to immobilize the molecule 3 having a desired specific binding ability on the carboxyl residue of the hydrophilic polymer. Furthermore, even after immobilization of the molecule 3, the titanium dioxide composite of the present invention can maintain a state of being uniformly dispersed in a wide range of PH regions including near neutrality due to the electric repulsion between the remaining carboxyl groups.
本発明において、 二酸化チタン複合体に分子識別能を付与する、 特異的な 結合能を有する分子 3としては、 目的分子と特異的に結合する分子であれば 下記に限定されるものではない。 このような分子間の特異的な結合は、 生体 内においては多種多様なものが明らかとなっている。 これらの中で、 最も重 要な分子としてタンパク質が挙げられる。 本発明によれば、 夕ンパク質とし て抗体、 リガンド、 レセプ夕一、 ポリ ■ 才リゴペプチド、 さらにはアミノ酸 まで好適に固定化が可能である。 また、 単純タンパク質の二酸化チタン複合 体への固定化にはァミノ基およびチオール基を、糖タンパク質の場合では糖 のアルデヒ ド基を、 固定化の際の標的官能基とすることが可能である。 また, 水溶性高分子によリ修飾した二酸化チ夕ンのカルボキシル基にビ才チン（ま たは、 アビジン） を導入しておき、 タンパク質をアビジン （または、 ピオチ ン） と粱橋させることによリ、 ビ才チン ： アビジンの相互作用を利用して固 定化することも可能である。 In the present invention, the molecule 3 having specific binding ability, which imparts molecular recognition ability to the titanium dioxide complex, is not limited to the following as long as it is a molecule that specifically binds to the target molecule. A wide variety of such specific bonds between molecules have been clarified in vivo. Of these, proteins are the most important molecules. According to the present invention, antibodies, ligands, receptor proteins, polysaccharide peptides, and even amino acids can be suitably immobilized as proteins. Also, titanium dioxide complex of simple protein For immobilization to the body, an amino group and a thiol group can be used, and in the case of a glycoprotein, an aldehyde group of a sugar can be used as a target functional group for immobilization. In addition, bi-tin (or avidin) is introduced into the carboxyl group of titanium dioxide modified with a water-soluble polymer, and the protein is allowed to crosslink with avidin (or piotin). It is also possible to immobilize it using the interaction of bijin: avidin.
さらに、本発明の二酸化チタン複合体は粒子表面に特定の因子やリガンド を提示することが可能である。 したがって、 例えば特定の受容体を発現して いるガン細胞のような細胞に対して、 リガンド ：受容体の特異的な結合によ り本複合体を細胞に導入することが可能である。 これらの因子やリガンドと しては、 上皮成長因子 （ E G F )、 トランスフォーミング増殖因子、 血小板 由来増殖因子、 骨形成因子、 神経成長因子等の増殖，成長因子や形成因子の 他に、 インターフェロン、 インターロイキン、 コロニー刺激因子、 腫瘍壊死 因子、 エリスロポエチン、 F a s抗原、 ァクチビン等のホルモンやリガンド 等が挙げられる。 これらのタンパク質も上記と同様に固定化が可能である。 すなわち、 これらに対応する受容体を特異的に発現している特定細胞に対し て、 ターゲティング可能なミサイルドラッグを構築することが可能となる。 近年、特定のタンパク質と特異的に結合する核酸アブ夕マーが注目されて いる。 このようなアブ夕マーも、 本発明の分子識別能を付与する特異的な結 合性を有する分子 3として利用が可能である。核酸の固定化を行う場合には, ポリメラーゼ チェーン リアクション （ P C R ) による D N A増幅の際に、 アミノ化プライマー、 ピオチン化プライマー、 チオール化プライマ一を用い て修飾 D N Aを合成することによリ、同様の方法で修飾二酸化チタン上へ固 定化することが可能である。 例えば、 アミノ化 D N Aを固定化に用いる場合. あらかじめ修飾二酸化チタンのカルボキシル基に N—ヒ ドロキシこはく酸 イミ ド （N H S ) のようなエステルを導入し、 求核置換反応によリアミノ化Furthermore, the titanium dioxide complex of the present invention can present a specific factor or ligand on the particle surface. Therefore, for example, it is possible to introduce the complex into a cell such as a cancer cell expressing a specific receptor by specifically binding the ligand: receptor. These factors and ligands include growth factors such as epidermal growth factor (EGF), transforming growth factor, platelet-derived growth factor, bone morphogenesis, nerve growth factor, etc., as well as interferon and interferon. Examples include hormones and ligands such as leukin, colony stimulating factor, tumor necrosis factor, erythropoietin, Fas antigen, and activin. These proteins can be immobilized as described above. That is, it becomes possible to construct a missile drug that can be targeted to a specific cell that specifically expresses a receptor corresponding thereto. In recent years, attention has been focused on nucleic acid abmers that specifically bind to specific proteins. Such an absormer can also be used as the molecule 3 having specific binding property that imparts the molecular discrimination ability of the present invention. When immobilizing nucleic acids, the same procedure is performed by synthesizing modified DNA using an aminated primer, a biotinylated primer, and a thiolated primer during DNA amplification by polymerase chain reaction (PCR). It can be immobilized on the modified titanium dioxide by a method. For example, when aminated DNA is used for immobilization. An ester such as N-hydroxysuccinic acid imide (NHS) is introduced into the carboxyl group of the modified titanium dioxide in advance, and then re-aminated by nucleophilic substitution.
D Aを修飾二酸化チ夕ンへ共有結合させることが可能である。チオール化 D Aを用いる場合も、 カルボキシル基に N H Sを反応させた後に 2 — （ 2 一ピリジニルジチ才） エタンァミンを作用させることにより、 同様にチ才 ール化 D N Aを修飾二酸化チタン上へ固定化することが可能である。 DA can be covalently linked to the modified titanium dioxide. Similarly, when thiolated DA is used, the thiolated DNA is immobilized on the modified titanium dioxide by reacting the carboxyl group with NHS and then reacting with 2- (2-pyridinyldithiethane) ethaneamine. Is possible.
被固定化分子のアルデヒ ド基を用いる場合は、カルボキシル基に N H Sを 反応させた後に、 ヒ ドラジンを用いることにより被固定化分子を修飾二酸化 チタンに結合し、 シァノホウ素化ナトリウムで還元すれば良い。 この他、 ビ 才チンヒ ドラジドゃァミノ化ビ才チンを用いてカルボキシル基をビ才チン 化させておけば、容易にァビジン化した被固定化分子を修飾二酸化チタン上 に導入できる。 このように適宜、 試薬、 修飾および架橋の方法を選択すれば. 修飾二酸化チタン上に導入したカルボキシル残基に多種多様な特異的な結 合能を有する分子 3を、 容易に固定化することが可能である。  When the aldehyde group of the molecule to be immobilized is used, the carboxyl group can be reacted with NHS, and then the molecule to be immobilized can be bound to the modified titanium dioxide by using hydrazine and reduced with sodium cyanoborohydride. . In addition, if the carboxyl group is converted into a bifunctional group by using a bifunctional hydrazide diamino-modified bitin, the avidin-modified molecule to be immobilized can be easily introduced onto the modified titanium dioxide. Thus, by appropriately selecting the reagents, modification and cross-linking methods, it is possible to easily immobilize a wide variety of molecules 3 having specific binding ability to the carboxyl residue introduced on the modified titanium dioxide. It is possible.
以上のように、修飾二酸化チタン上に導入したカルボキシル残基と結合可 能な官能基を有し、 かつその結合手法が明らかであれば、 特異的な結合能を 有する分子 （ 3 ) としてタンパク質や核酸あるいは糖類の他にも、 脂質や各 種生理活性物質等を好適に利用することが可能である。  As described above, if a functional group capable of binding to the carboxyl residue introduced on the modified titanium dioxide and the binding method is clear, a protein or a molecule having specific binding ability (3) can be used. In addition to nucleic acids or saccharides, lipids and various physiologically active substances can be suitably used.
一方、本発明の分子識別能を有する二酸化チタン複合体を水系の有害物質 処理や、医薬あるいは医療に応用しょうとする場合では生体内の生理的条件 から、 中性の水系溶媒に均一に分散していることが要求される。 上述したよ うに、本発明の分子識別能を有する二酸化チタン複合体は残余のカルボキシ ル残基を有しているため、水系溶媒中ではカルボキシル基の負電荷に由柰す る斥力が複合体間に作用する。 そのため、 p H 3〜 1 3の広範囲の p H領域 における水溶液中でも、本複合体は凝集することなく均一に分散した状態を 維持することが可能である。 したがって、 本発明の分子識別能を有する二酸 化チタン複合体を水、 種々の p H緩衢液、 輸液、 あるいは生理食塩水に分散 させた、 均一で安定な分散液を提供することが可能となる。 また、 本分散液 を含む軟 Θやスプレー剤等も製造が可能である。 この特性は、 特に二酸化チ タンを体内外の D D Sに応用する際に極めて有用である。 すなわち、 本 f 明 の分子識別能を有する二酸化チタン複合体の分散液は中性付近の生理的条 件においても凝集することがないために、患部組織に直接注射したリ静脈に 注射してターゲティングを行うことが可能となる。 また、 本分散液を含む軟 膏ゃスプレー剤を皮膚等の患部に直接塗布し、太陽光や紫外線ランプ等によ リ光治療を施すことが可能となる。 On the other hand, when the titanium dioxide complex having molecular recognition ability of the present invention is to be applied to aqueous harmful substance treatment, medicine or medical treatment, it is uniformly dispersed in a neutral aqueous solvent due to physiological conditions in a living body. Is required. As described above, since the titanium dioxide composite having molecular recognition ability of the present invention has a residual carboxy residue, the repulsive force due to the negative charge of the carboxyl group in the aqueous solvent is between the composites. Act on. Therefore, a wide pH range of pH 3 to 13 In the aqueous solution of the above, the composite can maintain a uniformly dispersed state without aggregation. Therefore, it is possible to provide a uniform and stable dispersion in which the titanium dioxide complex having molecular recognition ability of the present invention is dispersed in water, various pH buffer solutions, infusion solutions, or physiological saline. It becomes. In addition, a softener or a spray containing the present dispersion can also be produced. This property is especially useful when applying titanium dioxide to DDS inside and outside the body. That is, since the dispersion of the titanium dioxide complex having molecular recognition ability of the present invention does not aggregate under physiological conditions near neutrality, it is injected into the vein directly injected into the affected tissue and targeted. Can be performed. In addition, it becomes possible to apply an ointment / spray containing the present dispersion liquid directly to an affected area such as the skin, and to perform light treatment using sunlight or an ultraviolet lamp.
さらに、本発明の分子識別能を有する二酸化チタン複合体は単独で D D S として利用可能であることは無論のこと、 D D Sの一形態として他のキヤリ ァ中に包括させて利用することも可能である。この場合のキャリアとしては 特に制限はないが、 リボソーム、 ウィルス粒子、 中空ナノ粒子等を好適に用 いることができる。  Furthermore, it goes without saying that the titanium dioxide complex having molecular recognition ability of the present invention can be used alone as DDS, and it can also be used as a form of DDS in other carriers. . The carrier in this case is not particularly limited, but ribosomes, virus particles, hollow nanoparticles and the like can be suitably used.
本発明の分子識別能を有する二酸化チタン複合体を励起、活性化させるた めの光源装置は特別である必要はないが、二酸化チタンのバンドギヤップの 関係上その波長は 4 0 0 n m以下であることが望ましい。皮膚等の外用用途 では、 太陽光や通常の紫外線ランプ、 あるいはブラックライ 卜を好適に使用 できる。 また、 体内の患部に対しては内視鏡に紫外線ファイバーを装着する ことによリ紫外線を照射すれば良い。 さらに、 特に 2 8 0 n m付近の紫外線 を局所的に患部に照射して病変部を破壊しようとする光瘵法を想定した場 合では、その作用増強剤として本発明の分子識別能を有する二酸化チタン複 合体を適用することも可能である。 The light source device for exciting and activating the titanium dioxide composite having molecular discriminating ability of the present invention does not need to be special, but its wavelength is 400 nm or less due to the band gap of titanium dioxide. It is desirable. For external applications such as skin, sunlight, a normal ultraviolet lamp, or a black light can be suitably used. In addition, the affected part of the body may be irradiated with ultraviolet rays by attaching an ultraviolet fiber to the endoscope. In addition, especially when a photometric method of irradiating the affected area with ultraviolet rays near 280 nm locally to destroy the affected area is assumed, the carbon dioxide having the molecular discriminating ability of the present invention as an action enhancer is considered. Titanium compound It is also possible to apply coalescence.
以下に、 本発明を実施例に従って詳細に説明する。 ただし、 本発明はこの 実施例に制限されるものではない。  Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited to this embodiment.
(実施例 1 )  (Example 1)
二酸化チタン粒子へのポリアクリル醭の導入  Introduction of polyacrylic acid into titanium dioxide particles
チタンテ卜ライソプロボキシド 3. 6 gとイソプロパノール 3. 6 g 混 合し、 氷冷下で 6 0 m I の超純水に滴下して加水分解を行った。 滴下後に室 温で 3 0分間攪拌した。 攪拌後、 1 2 N硝酸を 1 m I 滴下して、 8 0 °Cで 8 時間攪拌を行い、 ぺプチゼーシヨンした。 ぺプチゼーシヨン終了後、 0. 4 5 c mのフィルターで濾過し、 脱塩カラム （ P D 1 0 ； アマシャム フアル マシア バイオサイエンス社） を用いて溶液交換して固形成分 1 %のアナ夕 一ゼ型ニ酸化チタンゾルを調製した。 この分散液を 1 0 0 m I 容のバイアル 瓶に入れ、 2 0 0 H zで 3 0分間超音波処理を行った。 超音波処理を行う前 と後での平均分散粒経はそれぞれ、 3 6. 4 n m、 2 0. 2 n mであった。 超音波処理後、 溶液を濃縮して固形成分 2 0 %の二酸化チタンゾル （アナ夕 ーゼ型） を調製した。  3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and hydrolyzed by dripping into 60 ml of ultrapure water under ice cooling. After the dropwise addition, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 N nitric acid (1 ml) was added dropwise, and the mixture was stirred at 80 ° C for 8 hours, followed by peptization.終了 After the completion of the filtration, the solution is filtered through a 0.45 cm filter, and the solution is exchanged using a desalting column (PD10; Amersham Pharmacia Biosciences) to convert the solid component into 1% anabolic nitric oxide. A titanium sol was prepared. This dispersion was placed in a vial of 100 ml in volume and subjected to ultrasonic treatment at 200 Hz for 30 minutes. The average particle size before and after the sonication was 36.4 nm and 20.2 nm, respectively. After the sonication, the solution was concentrated to prepare a titanium dioxide sol (analyse type) having a solid component of 20%.
得られた二酸化チタンゾル 0. 7 5 m I を 2 0 m I のジメチルホルムアミ ド （ D M F ) に分散させ、 ポリアクリル酸 （平均分子量 ： 5 0 0 0、 和光純 薬） 0. 2 gを溶解した D M Fを 1 O m l 添加後、 攪拌して混合した。 水熱 反応容器に溶液を移し変え、 1 8 0 °Cで 6時間水熱合成を行った。 反応終了 後、 反応容器温度が 5 0 °C以下になるまで冷却し、 溶液を取り出した後に水 8 0 m l を添加して攪拌混合した。エバポレー夕で D M Fおよび水を除去し た後に、 再度、 水 2 0 m I を添加してポリアクリル醆修飾二 H匕チタン水溶 液とした。 2 N塩酸 1 m I を添加して二酸化チタン粒子を沈殿させて、 遠心 後に上清を除去することにより未反応のポリァクリル酸を分離した。再度水 を添加して洗浄を行い、 遠心後に水を除去した。 5 0 m Mリン酸緩衝液 （ p0.75 mI of the obtained titanium dioxide sol is dispersed in 20 mI of dimethylformamide (DMF), and 0.2 g of polyacrylic acid (average molecular weight: 500, Wako Pure Chemical) is dissolved. After adding 1 O ml of the DMF, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel and hydrothermal synthesis was performed at 180 ° C for 6 hours. After the completion of the reaction, the reaction vessel was cooled to a temperature of 50 ° C. or lower. After taking out the solution, 80 ml of water was added and mixed with stirring. After removing DMF and water by evaporating, 20 ml of water was added again to obtain a polyacryl 醆 -modified dihydrogen titanium aqueous solution. Add 1 ml of 2 N hydrochloric acid to precipitate titanium dioxide particles, and centrifuge Thereafter, unreacted polyacrylic acid was separated by removing the supernatant. Water was added again for washing, and water was removed after centrifugation. 50 mM phosphate buffer (p
H 7 . 0 ) を 1 0 m I 添加後、 2 0 0 H 2で 3 0分間超音波処理を行い、 二 化チタン 子を分散させた。 超音波処理後、 0. 4 5 ^ mのフィル夕一で 過し、 固形成分 1 . 5 %のポリアクリル酸修飾二 S化チタンゾルを得た。 作製したポリァクリル酸修飾二酸化チタン微粒子 （アナ夕ーゼ型） の分散粒 径を測定したところ、 4 5 . 5 n mであった。 H 7.0) was added with 10 ml, and then ultrasonic treatment was performed with 200 H 2 for 30 minutes to disperse titanium dioxide. After the ultrasonic treatment, the mixture was passed through a 0.45 m filter to obtain a polyacrylic acid-modified titanium disodium sol having a solid content of 1.5%. The dispersion particle size of the prepared polyacrylic acid-modified titanium dioxide fine particles (analusase type) was measured to be 45.5 nm.
(実施例 2 )  (Example 2)
ポリアクリル酸修飾二酸化チタン微粒子への抗 A F P抗体分子の固定化 実施例 1 により得たポリアク リル酸修飾二酸化チタンゾル （アナターゼ 型） 1 m I を脱塩カラム P D 1 0を用いて溶液交換を行い、 水に分散したポ リアクリル酸修飾二酸化チタンゾル 3 m I を得た。 この溶液 1 . 5 m I に、 2 0 0 1\/1の 1 —ェチル— 3 — （ 3 —ジメチルァミノプロピル） カルボジィ ミ ドと 5 0 m Mの N—ヒ ドロキシこはく酸イミ ド （N H S ) の混合液 0. 1 m l を添加して 1 0分間攪拌を行い、 カルボキシル基を活性化した。 攪拌 終了後、 .1 0 m M酢酸緩衝液 （ p H 5 . 0 ) で平衡化した P D 1 0を用いて 溶液交換し、 1 0 m M酢酸緩衝液 （ p H 5 . 0 ) に分散したカルボキシル基 活性化ポリアクリル酸修飾二酸化チタンゾル 3 m I を得た。同一の緩衝液で 調製した抗 α—フェ 卜プロテイン (抗 A F P ) ポリクローナル抗体 (ャギ I g G、 S C - 8 1 0 8 ； コスモバイ才社） を 0. 0 5 m gノ m l になるよう に添加した。 室温で〗 5分間攪拌後、 0. 5 Mになるようにエタノールアミ ン塩酸塩水溶液 （ p H 8. 5 ) を添加した。 1 0分間攪拌後、 2 N塩酸を 1 m I 添加して二酸化チ夕ン粒子を沈殿させ、 違心後に上清を除去した。 再度 水を添加して洗浄を行い、遠心後に水を除去した。 5 0 m Mリン酸緩衝液（ p H 7. 0 ) を 2. 5 m l添加した後、 2 0 0 H zで 3 0分間超音波処理を行 い、 二酸化チタン粒子を分散させた。 超音波処理後、 0. 4 5 ju_ mのフィル 夕一で濾過し、 固形成分 0. 3 %の抗 A F P抗体固定化二酸化チタン複合体 ゾルとした。 作 ¾した抗 A F P抗体固定化二醸化チタン複合体 （アナターゼ 型） の分散粒? 測定したところ、 5 2 . 8 n mであった。 Immobilization of anti-AFP antibody molecules on polyacrylic acid-modified titanium dioxide fine particles 1 ml of the polyacrylic acid-modified titanium dioxide sol (anatase type) obtained in Example 1 was exchanged using a desalting column PD10, and the solution was exchanged. 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water was obtained. In 1.5 ml of this solution, 200 1 \ / 1 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinate imide (NHS ) Was added and stirred for 10 minutes to activate carboxyl groups. After completion of the stirring, the solution was exchanged using PD 10 equilibrated with a 10 mM acetate buffer (pH 5.0), and dispersed in a 10 mM acetate buffer (pH 5.0). A carboxyl group-activated polyacrylic acid-modified titanium dioxide sol 3 mI was obtained. Add an anti-α-fetoprotein (anti-AFP) polyclonal antibody (goat IgG, SC-810; CosmoBai Saisha) prepared in the same buffer to a concentration of 0.05 mg / ml. did. After stirring at room temperature for about 5 minutes, an aqueous solution of ethanolamine hydrochloride (pH 8.5) was added to a concentration of 0.5 M. After stirring for 10 minutes, 1 ml of 2N hydrochloric acid was added to precipitate titanium dioxide particles, and after eccentricity, the supernatant was removed. Water was again added for washing, and water was removed after centrifugation. 50 mM phosphate buffer (p After adding 2.5 ml of H7.0), ultrasonic treatment was performed at 200 Hz for 30 minutes to disperse the titanium dioxide particles. After the ultrasonic treatment, the solution was filtered through a filter of 0.45 jum in the evening to obtain a titanium dioxide composite sol having immobilized thereon an anti-AFP antibody having a solid content of 0.3%. The dispersion particle size of the prepared anti-AFP antibody-immobilized titanium dioxide complex (anatase type) was measured to be 52.8 nm.
(実施例 3 )  (Example 3)
ポリアクリル酸修飾二酸化チタン微粒子への抗 H S A抗体分子の固定化 実施例 1 により得たポリアク リル酸修飾二酸化チタンゾル （アナ夕ーゼ 型） 1 m I を脱塩カラム P D 1 0を用いて溶液交換を行い、 水に分散したポ リアクリル酸修飾二酸化チタンゾル 3 m I を得た。 この溶液 1 . 5 m l に 2 O O m Mの 1 —ェチル— 3 — （ 3—ジメチルァミノプロピル） カルボジィ ミ ドと 5 0 m Mの N—ヒ ドロキシこはく酸イミ ド （ N H S ) の混合液 0. 1 m I を添加して 1 0分間攪拌を行い、 カルボキシル基を活性化した。 攪拌 終了後、 1 0 m M酢酸緩衝液 （ p H 5 . 0 ) で平衡化した P D 1 0を用いて 溶液交換し、 1 0 m M酢酸緩衝液 （ p H 5 . 0 ) に分散したカルボキシル基 活性化ポリアクリル酸修飾二酸化チタンゾル 3 m I を得た。同一の緩衝液で 調製した抗ヒ 卜血清アルブミン （抗 H S A) モノクローナル抗体 （マウス I g G、 M S U— 3 0 4 ； コスモバイオ社） を 0. 0 5 m g /m l になるよう に添加した。 室温で 1 5分間攪拌後、 0. 5 Mになるようにエタノールアミ ン塩酸塩水溶液 （ p H 8. 5 ) を添加した。 1 0分間攪拌後、 2. 5 Mの N a C I 、 2 0 % ( w/ v ) ポリエチレングリコールを等量添加し二酸化チタ ン粒子を沈殿させ、 違心後に上清を除去した。 再度水を添加して洗浄を行い 違心後に水を除去した。 P B S緩衝液 （ p H 7. 0 ： 1 0 0 m Mの N a C I を含む、 日本ジーン） を 2. 5 m I 添加し、 二酸化チタン粒子を分散させた, 0. 4 5 z mのフィルターで濾過し、 固形成分 0. 3 %の抗 H S A抗体固定 化二酸化チタン複合体ゾルとした。作製した抗 H S A抗体固定化二酸化チタ ン複合体 （アナ夕一ゼ型） の分散粒徑を測定したところ、 5 2. 8 n mであ つた。 Immobilization of anti-HSA antibody molecules on polyacrylic acid-modified titanium dioxide fine particles 1 ml of polyacrylic acid-modified titanium dioxide sol (anasease type) obtained in Example 1 was exchanged using a desalting column PD 10 Was carried out to obtain 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water. In 1.5 ml of this solution, a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 50 mM N-hydroxysuccinic imid (NHS) 0 1 ml was added and the mixture was stirred for 10 minutes to activate the carboxyl group. After the stirring, the solution was exchanged using PD 10 equilibrated with 10 mM acetate buffer (pH 5.0), and the carboxyl dispersed in 10 mM acetate buffer (pH 5.0) was exchanged. A group-activated polyacrylic acid-modified titanium dioxide sol 3 mI was obtained. An anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG, MSU-304; Cosmo Bio) prepared in the same buffer was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 15 minutes, an aqueous solution of ethanolamine hydrochloride (pH 8.5) was added to a concentration of 0.5 M. After stirring for 10 minutes, equal amounts of 2.5 M NaCI and 20% (w / v) polyethylene glycol were added to precipitate titanium dioxide particles, and the supernatant was removed after eccentricity. Water was added again for washing, and water was removed after eccentricity. 2.5 ml of PBS buffer (pH 7.0: Nippon Gene containing 100 mM NaCI) was added to disperse the titanium dioxide particles, The mixture was filtered through a 0.45 zm filter to obtain a titanium dioxide composite sol having immobilized anti-HSA antibody having a solid content of 0.3%. The dispersion particle diameter of the prepared anti-HSA antibody-immobilized titanium dioxide complex (analyte type) was 52.8 nm.
(実施例 4 )  (Example 4)
ポリアク リル醆修飾二酸化チタン微粒子へのス トレプ卜ァビジン分子の固 定化 Immobilization of streptavidin molecules on polyacryl 醆 -modified titanium dioxide microparticles
実施例 1 により得たポリアクリル酸修飾二酸化チタンゾル （アナ夕ーゼ 型） 1 m I を脱塩カラム P D 1 0を用いて溶液交換を行い、 水に分散したポ リアクリル酸修飾二酸化チタンゾル 3 m I を得た。 この溶液 1 . 5 m l に 2 0 0 m Mの 1 ーェチル— 3— （ 3—ジメチルアミノプロピル） 力ルボジィミ ドと 5 0 m Mの N—ヒ ドロキシこはく酸イミ ド （N H S ) の混合液 0. 1 m I を添加して 1 0分間攪袢を行い、 カルボキシル基を活性化した。 攪拌終了 後、 1 0 m M酢酸緩衝液 （ p H 5. 0 ) で平衡化した P D 1 0を用いて溶液 交換し、 1 0 m M酢酸緩衝液 （ p H 5. 0 ) に分散したカルボキシル基活性 化ポリアクリル酸修飾二酸化チタンゾル 3 m I を得た。ス卜レブ卜アビジン ( P i e r c e B i o t e c h n o l o g y I n c . コード ： 2 1 1 2 6 ) を 0. 0 5 m g Zm I になるように添加した。 室温で 1 5分間攪拌後、 0. 5 Mになるようにエタノールァミン塩酸塩水溶液 （ p H 8. 5 ) を添加 した。 1 0分間攪拌後、 2. 5 Mの N a C I 、 Z O ^ Cw/ v) ポリェチレ ングリコールを等量添加し二酸化チタン粒子を沈殿させ、遠心後に上清を除 去した。 再度水を添加して洗浄を行い、 遠心後に水を除去した。 P B S緩衝 液 （ p H 7. 0、 日本ジーン） を 2. 5 m l 添加し、 二醆化チ夕ン粒子を分 散させた。 0. 4 5 μ ιτιのフィルターで濾過し、 固形成分 0. 3 %のス卜レ プ卜アビジン固定化二酸化チタン複合体ゾルとした。作製したス卜レプ卜ァ ビジン固定化二酸化チタン複合体 （アナ夕一ゼ型） の分散粒径を測定したと ころ、 5 0. 5 n mであった。 1 ml of the polyacrylic acid-modified titanium dioxide sol (anasease type) obtained in Example 1 was subjected to a solution exchange using a desalting column PD10, and 3 ml of a polyacrylic acid-modified titanium dioxide sol dispersed in water. Got. In 1.5 ml of this solution, a mixture of 200 mM 1-ethyl-3- (3-dimethylaminopropyl) sorbodimide and 50 mM N-hydroxysuccinate imide (NHS). 1 ml was added and the mixture was stirred for 10 minutes to activate the carboxyl group. After stirring, the solution was exchanged using PD 10 equilibrated with 10 mM acetate buffer (pH 5.0), and the carboxyl dispersed in 10 mM acetate buffer (pH 5.0) was exchanged. A group-activated polyacrylic acid-modified titanium dioxide sol 3 mI was obtained. Streptavidin (Pierce Biotechnology Inc. code: 211-26) was added to give 0.05 mg ZmI. After stirring at room temperature for 15 minutes, an aqueous solution of ethanolamine hydrochloride (pH 8.5) was added to a concentration of 0.5 M. After stirring for 10 minutes, an equal amount of 2.5 M NaCI and ZO ^ Cw / v) polyethylene glycol was added to precipitate titanium dioxide particles, and the supernatant was removed after centrifugation. Water was added again for washing, and water was removed after centrifugation. 2.5 ml of PBS buffer (pH 7.0, Nippon Gene) was added to disperse the distilled titanium particles. Filter through a 0.45 μιτι filter to obtain a 0.3% solid component. This was a putavidin-immobilized titanium dioxide composite sol. The dispersion particle size of the prepared streptavidin-immobilized titanium dioxide composite (ana-analyze type) was measured to be 50.5 nm.
(実施例 5 )  (Example 5)
ポリアクリル酸修飾磁性材複合二酸化チタン微粒子の合成  Synthesis of polyacrylic acid-modified magnetic material composite titanium dioxide fine particles
セパラプルフラスコ内にポリ才キシエチレン （ 1 5 ) セチルエーテル （C - 1 5 ； 日本サーファクタン卜工業社） を 4 5 . 1 6 を溶解させ、 5分間 窒素置換した後、 シクロへキセン溶液 （和光純薬） 7 5 m l を添加、 0. 6 7 Mの F e C I 2 (和光純薬） 水溶液 3. 6 m l を添加し、 2 5 0 r p mで 攪拌しながら、 3 0 %アンモニア水溶液 5 . 4 m l を添加し、 1 時間反応さ せた。 その後、 5 0 m Mテ卜ラエチルオルソシリケイ ト水溶液 (和光純薬） を 0. 4 m l 滴下し、 1 時間反応させた。 その後、 チタンテ卜ライソプロ ポキシド （和光純薬） を最終濃度 0. 0 0 5 Mになるように加えた。 5 0 % ( w/ V ) エタノール水溶液 1 0 m I を 1 m I ずつ 1 0分間隔で添加し た。 この反応液を遠心分離し、 沈殿物を 3 5 0 °Cで 2時間焼成した。 焼成後, 1 0 m M硝酸水溶液に分散させ、 超音波処理後、 0. 1 のフィルターで ろ過した。 得られた磁性材：複合二酸化チタンゾル 0. 7 5 m l を 2 0 m l のジメチルホルムアミ ド （ D M F ) に分散させ、 ポリアクリル酸 （平均分子 量 ： 5 0 0 0、 和光純薬） 0. 3 gを溶解した D M F 1 O m l を添加後、 攪 拌して混合した。 水熱反応容器 （ H U— 5 0、 三愛科学） に溶液を移し変え. 1 8 0 °Cで 6時間合成を行った。 反応終了後、 反応容器温度が 5 0 °C以下に なるまで冷却し、 分液漏斗に溶液を取り出した後、 水 1 O m l を添加して攪 拌混合した。 次いで、 クロ口ホル厶を 4 0 m I 加え攪拌混合して下層を除去 し、 上層を回収した。 このステップを 2回繰り返し、 D M Fを除去した。 こ の溶液 1 0 m l に 1 0 m l の 1 . 5 Mの N a C I 、 2 0 % (wZ v ) ポリエ チレングリコール 6 0 0 0 (和光純薬） を加え、 遠心後に上澄を除去した。 沈殿に 2 . 5 m l の水を加え、 S e p h a d e x G _ 2 5カラムにょリゲ ルろ過を行いポリアクリル續修飾磁性お'複合二酸化チタン徽粒子（アナター ゼ型） の分散液を得た。 45.16 was dissolved in polyseparable xylene (15) cetyl ether (C-15; Nippon Surfactant Industries, Ltd.) in a separable flask, and the atmosphere was replaced with nitrogen for 5 minutes. drugs) 7 added 5 ml, was added F e CI 2 (Wako pure Chemical) solution 3. 6 ml of 0. 6 7 M, with stirring at 2 5 0 rpm, 3 0% aqueous ammonia 5. 4 ml Was added and reacted for 1 hour. Thereafter, 0.4 ml of a 50 mM aqueous tetraethylorthosilicate solution (Wako Pure Chemical Industries, Ltd.) was added dropwise and reacted for 1 hour. Thereafter, titanium tetraisopropoxide (Wako Pure Chemical Industries) was added to a final concentration of 0.05 M. A 50% (w / V) aqueous solution of ethanol, 10 ml, was added in increments of 1 ml at 10 minute intervals. The reaction solution was centrifuged, and the precipitate was calcined at 350 ° C. for 2 hours. After baking, the mixture was dispersed in a 10 mM nitric acid aqueous solution, sonicated, and filtered with a 0.1 filter. Magnetic material obtained: 0.75 ml of composite titanium dioxide sol was dispersed in 20 ml of dimethylformamide (DMF), and polyacrylic acid (average molecular weight: 500, Wako Pure Chemical Industries) 0.3 After adding 1 ml of DMF in which g was dissolved, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel (HU-50, San-ai Science). Synthesis was performed at 180 ° C for 6 hours. After the completion of the reaction, the reaction vessel was cooled until the temperature of the reaction vessel became 50 ° C. or lower, and the solution was taken out into a separatory funnel. Next, 40 ml of black mouth was added, and the mixture was stirred and mixed to remove the lower layer, and the upper layer was recovered. This step was repeated twice to remove DMF. This To 10 ml of the above solution, 10 ml of 1.5 M NaCI, 20% (wZv) polyethylene glycol 600 (Wako Pure Chemical Industries) was added, and the supernatant was removed after centrifugation. To the precipitate was added 2.5 ml of water, and the mixture was filtered through a Sephadex G_25 column to obtain a dispersion of polyacrylic continuously modified magnetic and composite titanium dioxide particles (anatase type).
(実施例 6 )  (Example 6)
ポリアクリル酸修飾磁性材複合二酸化チ夕ン微粒子への抗 H S A抗体分 子の固定化  Immobilization of anti-HSA antibody molecule on polyacrylic acid-modified magnetic material composite carbon dioxide fine particles
実施例 5で得られたポリァクリル酸修飾磁性材複合二酸化チタン微粒子の 分散液 1 . 5 m l に 2 0 0 m Mの 1 一ェチル一 3 — ( 3—ジメチルアミノプ 口ピル） カルポジイミ ドと 5 O m Mの N—ヒ ドロキシこはく酸イミ ド （ N H S ) の混合液 0. 1 m l を添加して 1 0分間攪拌を行い、 カルボキシル基を 活性化した。 攪拌終了後、 1 0 m M酢酸緩衝液 （ p H 5 . 0 ) で平衡化した P D 1 0を用いて溶液交換し、 1 0 m M酢酸緩衝液 （ p H 5. 0 ) に分散し たカルボキシル基活性化ポリアクリル酸修飾磁性材複合二酸化チタンゾル 3 m l を得た。 同一の緩衝液で調製した抗ヒ 卜血清アルブミン （抗 H S A) モノクローナル抗体 （マウス I g G ： M S U— 3 0 4、 コスモバイオ社） を 0. 0 5 m g Zm I になるように添加した。 室温で 1 5分間攪拌後、 0. 5 Mになるようにエタノールアミン塩酸塩水溶液 （ p H 8. 5 ) を添加した。 In 1.5 ml of the dispersion of the polyacrylic acid-modified magnetic material composite titanium dioxide fine particles obtained in Example 5, 200 mM 1-ethyl-13- (3-dimethylaminopropyl) carbopimide and 5O A carboxyl group was activated by adding 0.1 ml of a mixture of mM N-hydroxysuccinic acid imid (NHS) and stirring for 10 minutes. After the stirring, the solution was exchanged using PD 10 equilibrated with 10 mM acetate buffer (pH 5.0), and dispersed in 10 mM acetate buffer (pH 5.0). 3 ml of a carboxyl group-activated polyacrylic acid-modified magnetic material composite titanium dioxide sol was obtained. An anti-human serum albumin (anti-HSA) monoclonal antibody (mouse IgG: MSU—304, Cosmo Bio Inc.) prepared in the same buffer was added to a concentration of 0.05 mg ZmI. After stirring at room temperature for 15 minutes, an aqueous solution of ethanolamine hydrochloride (pH 8.5) was added to a concentration of 0.5 M.
1 0分間攪拌後、 2. 5 Mの N a C I 、 2 0 % ( w/ v ) ポリエチレングリ コールを等量添加して磁性材複合二酸化チタン粒子を沈殿させ、違心後に上 清を除去した。 再度水を添加して洗浄を行い、 遠心後に水を除去した。 P B S (日本ジーン） を 2 . 5 m l 添加し、 磁性材複合二醱化チタン粒子を分散 させた。 0. 4 5 mのフィルターで濾過し、 固形成分 0. 3 %の抗 H S A 抗体固定化磁性材複合二酸化チタン複合体ゾルとした。作製した抗 H S A抗 体固定化磁性材複合二酸化チタン複合体 （アナターゼ型） の分散粒径を測定 したところ、 1 0 5 n mであった。 After stirring for 10 minutes, an equal amount of 2.5 M NaCI, 20% (w / v) polyethylene glycol was added to precipitate magnetic material composite titanium dioxide particles, and the supernatant was removed after eccentricity. Water was added again for washing, and water was removed after centrifugation. 2.5 ml of PBS (Nippon Gene) was added to disperse the magnetic composite titanium dioxide particles. Filter through a 0.45 m filter to obtain 0.3% solid HSA An antibody-immobilized magnetic material composite titanium dioxide composite sol was obtained. The dispersion particle size of the prepared anti-HSA antibody-immobilized magnetic material composite titanium dioxide composite (anatase type) was measured to be 105 nm.
(実施例 7 )  (Example 7)
二酸化チ夕ン粒子へのァクリル酸ノスルホン酸系共重合体の導入  Introduction of acrylic acid / nosulfonic acid copolymer into titanium dioxide particles.
実施例 1 で得られた固形成分 2 0 %の二酸化チ夕ンゾル （アナ夕ーゼ型） 0 . 7 5 m l を 2 0 m l のジメチルホル厶アミ ド （ D M F ) に分散させ、 ァ クリル酸/スルホン酸系モノマー共重合体 （平均分子量： 5 0 0 0、 プロ 卜 ン置換後凍結乾燥した標品、 日本触媒） 0 . 3 gを溶解した D M F 1 0 m I を添加後、 撹拌して混合した。 水熱反応容器 （H U — 5 0、 三愛科学） に溶 液を移し、 1 5 0 °Cで 5時間反応させた。 反応終了後、 反応容器を室温にな るまで冷却し、 反応液に対して 2倍量のイソプロパノール （和光純薬） を添 加した。 室温で 3 0分以上静置後、 4 0 0 0 X g、 2 0分間遠心分離を行い. 沈殿物を回収した。 この沈殿物を 7 0 %エタノールで洗浄後、 2 . 5 m l の 水を加えて、 アクリル酸ノスルホン酸系共重合体修飾二酸化チタンゾル （ァ ナ夕一ゼ型） を得た。  0.75 ml of 20% titanium dioxide sol (anasease type) of the solid component obtained in Example 1 was dispersed in 20 ml of dimethylformamide (DMF), and acrylic acid / sulfone was added. Acid-based monomer copolymer (average molecular weight: 500, sample freeze-dried after substitution with toluene, Nippon Shokubai) 0.3 g of DMF dissolved in 10 mI was added, followed by stirring and mixing. . The solution was transferred to a hydrothermal reaction vessel (HU-50, San-ai Science) and reacted at 150 ° C for 5 hours. After completion of the reaction, the reaction vessel was cooled to room temperature, and twice the amount of isopropanol (Wako Pure Chemical Industries) was added to the reaction solution. After leaving still at room temperature for 30 minutes or more, centrifugation was performed at 4000 X g for 20 minutes. The precipitate was collected. After washing this precipitate with 70% ethanol, 2.5 ml of water was added thereto to obtain an acrylic acid / nosulfonate-based copolymer-modified titanium dioxide sol (analysis type).
(実施例 8 )  (Example 8)
アクリル酸ノスルホン酸系共重合体修飾二酸化チタン微粒子への抗 D R 4抗体分子の固定化  Immobilization of anti-DR4 antibody molecules on acrylic acid / sulfonic acid-based copolymer-modified titanium dioxide fine particles
実施例 7で作製したァク リル酸/スルホン酸系共重合体修飾二酸化チタ ンゾル Ί . 5 m I に 2 0 O m Mの 1 —ェチル一 3 — ( 3—ジメチルアミノプ 口ピル） カルポジイミ ドと 5 O m Mの N —ヒ ドロキシこはく酸イミ ド （N H S ) の混合液 0 . 1 m I を添加して 1 0分間撹拌を行い、 カルボキシル基を 活性化した。 撹拌終了後、 1 O m M酢酸緩衝液 （ p H 5 . 0 ) で平衡化した P D 1 0を用いて溶液交換し、 1 0 m M酢酸緩衝液 （ p H 5. 0 ) に分散し たカルボキシル基活性化ァクリル酸/スルホン酸系共重合体修飾二酸化チ 夕ンゾル 3 m I を得た。 これに、 抗 D R 4モノクローナル抗体 （A n t i - T R A I L R e c e p t o r 1 、 マウス、 コード ： S A— 2 2 5、 フナコ シ社） を 0. 0 5 m g /m I になるように添加した。 室温で 1 分間撹拌後、 0. 5 Mになるようにエタノールアミン塩醆塩水溶液 （ p H 8. 5 ) を添加 した。 室温で 1 0分間撹袢後、 2. 5 Mの N a C I 、 2 0 % ( wZ v ) ポリ エチレングリコールを等量添加して二酸化チタン粒子を沈殿させ、遠心分離 によリ上清を除去した。 水で洗浄の後遠心分離して沈殿を回収し、 P B S緩 衝液 （ P H 7. 0、 日本ジーン） を 2. 5 m 忝加して二酸化チタン粒子を 分散させた。 0. 4 5 mのフィルターで濾過し、 固形成分 0. 3 %の抗 D R 4抗体固定化二酸化チタン複合体ゾル （アナターゼ型） を得た。 Acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol prepared in Example 7. 0.5 OmM / 20 OmM 1-ethyl-13- (3-dimethylaminopropyl) carbodiimide Then, 0.1 ml of a mixed solution of N-hydroxysuccinate imide (NHS) and 5 OmM of N-hydroxysuccinic acid was added, and the mixture was stirred for 10 minutes to activate the carboxyl group. After stirring, the mixture was equilibrated with 1 O mM acetate buffer (pH 5.0). The solution was exchanged using PD 10 and 3 ml of a carboxyl group-activated acrylic acid / sulfonic acid copolymer-modified titanium dioxide sol dispersed in 10 mM acetate buffer (pH 5.0) was added. Obtained. To this, an anti-DR4 monoclonal antibody (Anti-TRAILR eceptor 1, mouse, code: SA-225, Funakoshi) was added to a concentration of 0.05 mg / ml. After stirring at room temperature for 1 minute, an ethanolamine salt / salt aqueous solution (pH 8.5) was added to a concentration of 0.5 M. After stirring at room temperature for 10 minutes, an equal amount of 2.5 M NaCI and 20% (wZv) polyethylene glycol was added to precipitate titanium dioxide particles, and the supernatant was removed by centrifugation. did. After washing with water, the precipitate was recovered by centrifugation, and 2.5 μm of PBS buffer (PH 7.0, Nippon Gene) was added to disperse the titanium dioxide particles. The solution was filtered through a 0.45 m filter to obtain an anti-DR4 antibody-immobilized titanium dioxide complex sol (anatase type) having a solid content of 0.3%.
(実施例 9 )  (Example 9)
抗 A F P抗体固定化二酸化チタン複合体による抗原 A F Pの分解  Degradation of antigen AFP by anti-AFP antibody-immobilized titanium dioxide complex
α-フエ 卜プロテイン （A F P、 コスモバイオ社） を 1 At g Zm I になる ように 5 0 m Mの P B S緩衝液 （ p H 7 . 0、 日本ジーン） で希釈し、 実施 例 2で作製した抗 A F P抗体固定化二酸化チタン複合体を固形成分 0 · 0 1 %になるように添加した。 次いで、 3 7 °Cで 3時間静置して抗原抗体反応 による凝集体を形成させた。 A F Pと抗 A F P抗体固定化二酸化チタン複合 体が凝集体を形成したことから、抗 A F P抗体固定化二酸化チタン複合体が 特異的に A F Pを認識して結合していることは明らかである。 この凝集体を 攪拌しながら波長 3 4 0 n mの紫外光を 1 m W / c m 2 になるように照射 し、 6 0 0 n mにおける波長の吸収 （凝集体の謂度） を分光光度計により測 定した。 結果を図 2に示す。 紫外線 （ U V ) 照射時にのみ、 凝集体濃度の低 下にともなう吸光度の減少が認められる。 すなわち、 抗 A F P抗体固定化二 酸化チタン複合体の光触媒作用により、抗原 A F Pが分解されることが明ら かとなつた。 α-Fet protein (AFP, Cosmo Bio) was diluted with 50 mM PBS buffer (pH 7.0, Nippon Gene) to 1 Atg ZmI and prepared in Example 2. An anti-AFP antibody-immobilized titanium dioxide complex was added so that the solid content was 0.101%. Then, the mixture was allowed to stand at 37 ° C. for 3 hours to form an aggregate by an antigen-antibody reaction. The formation of the aggregate of the AFP and the titanium dioxide complex immobilized with the anti-AFP antibody clearly indicates that the anti-AFP antibody immobilized titanium dioxide complex specifically recognizes and binds to AFP. The ultraviolet light having a wavelength of 3 4 0 nm while stirring the aggregate is irradiated so that the 1 m W / cm 2, the absorption wavelength (Ido aggregate) in 6 0 0 nm measured by a spectrophotometer Specified. The result is shown in figure 2. Low aggregate concentration only during ultraviolet (UV) irradiation A decrease in absorbance is observed below. That is, it became clear that the antigen AFP was degraded by the photocatalysis of the titanium dioxide complex immobilized with the anti-AFP antibody.
(実施例 1 0 )  (Example 10)
抗 H S A抗体固定化二酸化チ夕ン複合体による抗原一抗体反応の確認 ヒ 卜血清アルプミン （ H S A、 コスモバイ才社） € 2 5 0 g /m l にな るように 5 0 m Mの P B S緩衝液 （ p H 7 . 0、 日本ジーン） で希釈した。 別途、 4 0 0 ^11^の 1 —ェチルー 3 — ( 3—ジメチルァミノプロピル） カル ポジイミ ドと 1 O O m Mの N—ヒ ドロキシこはく酸イミ ド （N H S ) の混合 液で表面プラズモン共鳴センサのセンサチップ C 1 ( B I A C O R E社） を 活性化した。 このセンサチップを表面プラズモン共鳴測定装置： B I A C O R E 1 0 0 0 ( B I A C O R E社） に装着し、 先の H S A溶液を流速 1 0 n I /m i nで通液した後、 0. 1 Mエタノールァミンにより活性基のプロッ キングを行い、 H S A固定化センサチップを作製した。 この H S A固定化セ ンサチップへ実施例 3で作製した 0. 0 1 %の抗 H S A抗体固定化二酸化チ タン複合体ゾル、 および実施例 4で作製した 0. 0 1 %のス卜レプ卜ァビジ ン固定化二酸化チタン複合体ゾルを送液して抗原—抗体反応を確認した。結 果を図 3に示す。 H S A固定化センサチップに対し、 抗 H S A抗体固定化二 酸化チタン複合体は反応してチップに結合しているが、ス卜レプ卜ァビジン 固定化二酸化チタン複合体は反応せず結合は起こらなかった。 すなわち、 二 酸化チタン上の親水性高分子に固定化された抗 H S Aモノクローナル抗体 は、 固定化後も抗体としての活性を確実に保持していることが確認された。 (実施例 1 1 )  Confirmation of antigen-antibody reaction with anti-HSA antibody-immobilized titanium dioxide complex Human serum albumin (HSA, CosmoBai Co., Ltd.) 50 mM PBS buffer (250 mM / ml) pH 7.0, Nippon Gene). Separately, a surface plasmon resonance sensor using a mixed solution of 1-ethyl 3- (3-dimethylaminopropyl) carbodiimide of 400 ^ 11 ^ and N-hydroxysuccinate imide (NHS) of 100 M M Sensor chip C 1 (BIACORE) was activated. This sensor chip was attached to a surface plasmon resonance measurement device: BIACORE 100 (BIACORE), and the HSA solution was passed at a flow rate of 10 nI / min, and then activated with 0.1 M ethanolamine. HSA was immobilized to produce an HSA-immobilized sensor chip. To this HSA-immobilized sensor chip, 0.1% of anti-HSA antibody-immobilized titanium dioxide complex sol prepared in Example 3 and 0.01% of streptavidin prepared in Example 4 The immobilized titanium dioxide complex sol was sent to confirm the antigen-antibody reaction. The results are shown in Figure 3. The anti-HSA antibody-immobilized titanium dioxide complex reacted with the HSA-immobilized sensor chip and bound to the chip, but the streptavidin-immobilized titanium dioxide complex did not react and did not bind. . That is, it was confirmed that the anti-HSA monoclonal antibody immobilized on the hydrophilic polymer on titanium dioxide certainly retained the activity as an antibody even after immobilization. (Example 11)
抗 H S A抗体固定化二酸化チタン複合体による抗原 H S Aの分解 H S Aを 2 0 n g /m l になるように P B S緩衝液 （ p H 7 . 0、 日本ジ ーン） で希釈し、 実施例 3で作製した抗 H S A抗体固定化二酸化チタン複合 体を固形成分 0 . 0 1 %になるように添加した。 次いで、 室温で 3 0分放置 後、 波長 3 4 0 n mの紫外光を 1 m W / c m 2 になるように照射し、 1 5分 毎にサンプリングを 9 0分間行った。実施例 4で作製したストレプ卜ァビジ ン固定化二酸化チタン複合体についても同様の処理を行った。 別途、 実施例 8の方法に準じて、表面プラズモン共鳴測定用の抗 H S Aポリクローナル抗 体 （ゥサギ） 固定化センサチップを作製した。 実施例 8と同様に B I A C 0 R E 1 0 0 0を用い、各経時サンプルを抗 H S A抗体固定化センサチップに 2 0 μ. \ 送液し、 次いで 2次抗体として抗 H S Aポリクローナル抗体 （ゥサ ギ） 5 0 /z g /m I を 1 I 送液してサンドイッチアツセィを行い、 抗体 送液後 1 0秒後の R U値 （結合量に相当） を測定した。 U V未照射時の R U 値を 1 0 0 %とした相対値から算出した H S Αの分解率を図 4に示す。図 4 の結果から、抗 H S A抗体固定化二酸化チタン複合体はス卜レブ卜アビジン 固定化二酸化チタン複合体と比べ、 H S Aの分解速度が極めて速いことが示 された。 産業上の利用可能性 Degradation of antigen HSA by anti-HSA antibody immobilized titanium dioxide complex HSA was diluted to a concentration of 20 ng / ml with a PBS buffer (pH 7.0, Nippon Gene), and the anti-HSA antibody-immobilized titanium dioxide complex prepared in Example 3 was added to a solid component of 0.1%. It was added so as to be 0 1%. Next, after standing at room temperature for 30 minutes, ultraviolet light having a wavelength of 340 nm was irradiated so as to have a wavelength of 1 mW / cm 2 , and sampling was performed every 15 minutes for 90 minutes. The same treatment was performed on the streptavidin-immobilized titanium dioxide composite prepared in Example 4. Separately, according to the method of Example 8, an anti-HSA polyclonal antibody (Egret) immobilized sensor chip for surface plasmon resonance measurement was prepared. Using BIAC 0 RE1000 as in Example 8, each time-lapsed sample was sent to the anti-HSA antibody-immobilized sensor chip at 20 μ. \, And then the anti-HSA polyclonal antibody (Egret) was used as a secondary antibody. ) 50 / zg / mI was sent at 1 I, sandwich was performed, and the RU value (corresponding to the amount of binding) 10 seconds after the antibody was sent was measured. Figure 4 shows the decomposition rate of HS II calculated from the relative value when the RU value without UV irradiation was 100%. The results shown in FIG. 4 indicate that the anti-HSA antibody-immobilized titanium dioxide complex has a much higher HSA degradation rate than the streptavidin-immobilized titanium dioxide complex. Industrial applicability
本発明により、 内分泌撹乱物質、 病因分子、 ガン細胞等と特異的に結合し. かつ光触媒作用によりそれらの分解作用を示す、分子識別能を有する二酸化 チタン複合体を提供することができる。  According to the present invention, it is possible to provide a titanium dioxide complex having a molecular discriminating ability, which specifically binds to endocrine disrupting substances, pathogenic molecules, cancer cells, and the like, and exhibits a decomposing action thereof by photocatalysis.

Claims

請求の範囲 The scope of the claims
1 . 二酸化チタンの表面が、 カルボキシル基を有する親水性高分子により 修飾された二酸化チタン複合体であって、該親水性高分子の力 Jレボキシル基 と二酸化チタンがエステル結合で結合しているとともに、該親水性高分子の 力ルポキシル残基に、 目的分子に対して特異的な結合能を有する分子を固定 化したことを特徴とする、 分子識別能を有する二酸化チ夕ン複合体。 1. The surface of titanium dioxide is a titanium dioxide complex modified with a hydrophilic polymer having a carboxyl group, and the force of the hydrophilic polymer J reboxyl group and titanium dioxide are bonded by an ester bond. A titanium dioxide complex having a molecular discriminating ability, wherein a molecule having a specific binding ability to a target molecule is immobilized on a lipoxyl residue of the hydrophilic polymer.
2 . 前記二酸化チタンが、 アナターゼ型、 またはルチル型である、 請求項 1 に記載の分子識別能を有する二酸化チタン複合体。 2. The titanium dioxide composite having molecular recognition ability according to claim 1, wherein the titanium dioxide is an anatase type or a rutile type.
3 . 前記二酸化チタンの粒径が、 2 ~ 2 0 0 n mであることを特徴とする. 請求項 1 または 2に記載の分子識別能を有する二酸化チタン複合体。 3. The particle size of the titanium dioxide is 2 to 200 nm. 3. The titanium dioxide composite having molecular recognition ability according to claim 1 or 2.
4 . 前記二酸化チタンが、 二酸化チタンと磁性材とからなる複合二酸化チ タンであることを特徴とする、請求項 1 〜 3いずれか一項に記載の分子識別 能を有する二酸化チタン複合体。 4. The titanium dioxide composite having molecular discriminating ability according to any one of claims 1 to 3, wherein the titanium dioxide is a composite titanium dioxide comprising titanium dioxide and a magnetic material.
5 . 前記親水性高分子が、 水溶性高分子であることを特徴とする、 請求項 1 〜 4のいずれか一項に記載の分子識別能を有する二酸化チタン複合体。 5. The titanium dioxide composite having molecular recognition ability according to any one of claims 1 to 4, wherein the hydrophilic polymer is a water-soluble polymer.
6 . 前記水溶性高分子が、 ポリカルボン酸を含むことを特徴とする、 請求 項 5に記載の分子識別能を有する二酸化チタン複合体。 6. The titanium dioxide composite having molecular discriminating ability according to claim 5, wherein the water-soluble polymer contains a polycarboxylic acid.
7 . 前記水溶性高分子が、 分子中に複数のカルボキシル基単位を有する共 重合体を含むことを特徴とする、請求項 5に記載の分子識別能を有する二酸 化チタン複合体。 7. The titanium dioxide composite having molecular recognition ability according to claim 5, wherein the water-soluble polymer includes a copolymer having a plurality of carboxyl group units in the molecule.
8 . 前記目的分子に対して特異的な結合能を有する分子が、 アミノ酸、 ぺ プチド、 単純タンパク質、 複合タンパク質、 および抗体であることを特徴と する、請求項 1〜 7のいずれか一項に記載の分子識別能を有する二酸化チタ ン複合体。 8. The method according to any one of claims 1 to 7, wherein the molecule having a specific binding ability to the target molecule is an amino acid, a peptide, a simple protein, a complex protein, and an antibody. A titanium dioxide complex having the molecular recognition ability described in the above.
9 . 前記目的分子に対して特異的な結合能を有する分子が、 ヌクレオシド, ヌクレオチド、 核酸、 およびペプチド核酸であることを特徴とする、 請求項 1〜 7のいずれか一項に記載の分子識別能を有する二酸化チタン複合体。 9. The molecule having a specific binding ability to the target molecule is a nucleoside, a nucleotide, a nucleic acid, and a peptide nucleic acid, The molecule identification according to any one of claims 1 to 7, Titanium dioxide composite with function.
1 0 . 前記目的分子に対して特異的な結合能を有する分子が、 単糖、 糖鎖, 多糖、 および複合糖質であることを特徴とする、 請求項 1〜 7のいずれか一 項に記載の分子識別能を有する二酸化チタン複合体。 10. The molecule according to any one of claims 1 to 7, wherein the molecule having a specific binding ability to the target molecule is a monosaccharide, a sugar chain, a polysaccharide, and a complex saccharide. A titanium dioxide composite having the molecular discriminating ability as described above.
1 1 . 前記目的分子に対して特異的な結合能を有する分子が、 脂肪酸、 脂 肪酸誘導体、 単純脂質、 複合脂質であることを特徴とする、 請求項 1 ~ 7の いずれか一項に記載の分子識別能を有する二酸化チタン複合体。 11. The molecule according to any one of claims 1 to 7, wherein the molecule having a specific binding ability to the target molecule is a fatty acid, a fatty acid derivative, a simple lipid, or a complex lipid. A titanium dioxide composite having the molecular discriminating ability as described above.
1 2 . 前記目的分子に対して特異的な結合能を有する分子が、 生理活性物 質であることを特徵とする、請求項 1〜 7のいずれか一項に記載の分子識別 能を有する二酸化チタン複合体。 12. The molecule having a molecular discriminating ability according to any one of claims 1 to 7, wherein the molecule having a specific binding ability to the target molecule is a physiologically active substance. Titanium composite.
1 3 . 生体への導入が許容ざれる水溶液中に、 請求項 8〜 1 2のいずれか13. Any of claims 8 to 12 in an aqueous solution that is not acceptable for introduction into a living body.
—項に記載の分子識別能を有する二酸化チタン複合体を含むことを特徵と する、 分子識別能を有する二酸化チタン複合体の分散液。 —A dispersion liquid of a titanium dioxide complex having a molecular discriminating ability, which comprises the titanium dioxide complex having a molecular discriminating ability according to the above item.
1 4 . 前記水溶液が p H緩衢液であることを特徵とする、 請求項〗 3記載 の分子識別能を有する二酸化チタン複合体の分散液。 14. The dispersion of a titanium dioxide composite having molecular recognition ability according to claim 3, wherein the aqueous solution is a pH buffer.
1 5 . 前記水溶液が生 ί旱食塩水であることを特徴とする、 請求項 1 3記載 の分子識別能を有する二酸化チタン複合体の分散液。 15. The dispersion of a titanium dioxide composite having molecular recognition ability according to claim 13, wherein the aqueous solution is a fresh saline solution.
1 6 . 生体への導入が許容される包括体に、 該分子識別能を有する二酸化 チタン複合体が包括されていることを特徴とする、請求項 1 3〜 1 5のいず れか一項に記載の分子識別能を有する二酸化チタン複合体の分散液。 16. The package according to any one of claims 13 to 15, wherein the titanium dioxide complex having the molecular discriminating ability is included in a package that is allowed to be introduced into a living body. 4. A dispersion of a titanium dioxide composite having a molecular discriminating ability according to 1.).
1 7 . 前記包括体が、 リボソーム、 ゥィルス粒子、 中空ナノ粒子のいずれ かであることを特徴とする、請求項 1 6 に記載の分子識別能を有する二酸化 チタン複合体の分散液。 17. The dispersion liquid of a titanium dioxide composite having molecular recognition ability according to claim 16, wherein the inclusion body is any one of ribosome, virus particle, and hollow nanoparticle.
PCT/JP2004/004638 2003-03-31 2004-03-31 Titanium dioxide complex having molecule distinguishability WO2004087765A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP04724768A EP1614694A4 (en) 2003-03-31 2004-03-31 Titanium dioxide complex having molecule distinguishability
AU2004226053A AU2004226053A1 (en) 2003-03-31 2004-03-31 Titanium dioxide complex having molecule distinguishability
JP2005504274A JPWO2004087765A1 (en) 2003-03-31 2004-03-31 Titanium dioxide complex with molecular recognition ability
US10/551,164 US20060281087A1 (en) 2003-03-31 2004-03-31 Titanium dioxide complex having molecule distinguishability

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2003-94429 2003-03-31
JP2003094429 2003-03-31
JP2003-340234 2003-09-30
JP2003340234 2003-09-30

Publications (1)

Publication Number Publication Date
WO2004087765A1 true WO2004087765A1 (en) 2004-10-14

Family

ID=33134316

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/004638 WO2004087765A1 (en) 2003-03-31 2004-03-31 Titanium dioxide complex having molecule distinguishability

Country Status (5)

Country Link
US (1) US20060281087A1 (en)
EP (1) EP1614694A4 (en)
JP (1) JPWO2004087765A1 (en)
AU (1) AU2004226053A1 (en)
WO (1) WO2004087765A1 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005289660A (en) * 2004-03-31 2005-10-20 Toto Ltd Surface-modified titanium dioxide particulate and dispersion thereof and method for manufacturing the same
EP1779855A1 (en) * 2005-10-28 2007-05-02 Abdula Kurkayev Nanoparticles of a heterocrystal mineral for use as a medicament and method of producing the same
WO2007048635A1 (en) * 2005-10-28 2007-05-03 Abdula Kurkayev Method of activating a photosensitizer
WO2007122956A1 (en) * 2006-03-24 2007-11-01 Toto Ltd. Titanium oxide complex particle, dispersion solution of the particle, and process for production of the particle
JP2008535788A (en) * 2005-02-28 2008-09-04 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Long-lasting water-resistant sunscreen comprising a metal oxide and a peptide conditioner
WO2008111538A1 (en) * 2007-03-09 2008-09-18 Kazufumi Ogawa Magnetic-sensitive material, magnetic-sensitive medicinal agent, and those production methods
WO2009144775A1 (en) * 2008-05-29 2009-12-03 Toto株式会社 Anti-tumor agent
WO2010016581A1 (en) * 2008-08-08 2010-02-11 Toto株式会社 Ultrasonic cancer therapy accelerator
US7935329B2 (en) * 2005-12-20 2011-05-03 Lg Chem, Ltd. Titania sol, method of preparing the same, and coating composition comprising the same
WO2011102407A1 (en) 2010-02-17 2011-08-25 国立大学法人神戸大学 Radiation therapy agent
JP2017122684A (en) * 2016-01-08 2017-07-13 積水メディカル株式会社 Immunoagglutination particles and method for manufacturing the particles

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087577A1 (en) * 2003-03-31 2004-10-14 Toto Ltd. Surface-modified titanium dioxide fine particles and dispersion comprising the same, and method for producing the same
JP5044150B2 (en) * 2005-08-05 2012-10-10 Ｔｏｔｏ株式会社 Pharmaceutical titanium dioxide composite that loses its efficacy by light irradiation
WO2007034586A1 (en) * 2005-09-22 2007-03-29 Toto Ltd. Photocatalytic titanium dioxide microparticle, dispersion liquid thereof and process for producing the same
JP5757038B2 (en) * 2006-06-12 2015-07-29 キャプティゲル アクチエボラグ Metal oxide hydrogels and hydrosols, their manufacture and use
WO2009038236A1 (en) * 2007-09-20 2009-03-26 Fujifilm Corporation Air cleaning apparatus
EP2370503B1 (en) * 2008-12-03 2017-11-08 EcoSynthetix Ltd. Process for producing biopolymer nanoparticle biolatex compositions having enhanced performance and compositions based thereon
GB0921596D0 (en) 2009-12-09 2010-01-27 Isis Innovation Particles for the treatment of cancer in combination with radiotherapy
KR20120118065A (en) 2010-02-12 2012-10-25 디에스엠 아이피 어셋츠 비.브이. Single unit antibody purification
US8968789B2 (en) * 2013-02-13 2015-03-03 California Institute Of Technology Sulfonic esters of metal oxides and methods of their use
EP3094356A4 (en) 2014-01-17 2017-06-14 California Institute of Technology Sulfonic esters of metal oxides and methods of their use
KR101631371B1 (en) * 2014-07-18 2016-06-17 경북대학교 산학협력단 A biochip comprising covalently immobilized bioactive molecules through organic couplers thereon
JP6654871B2 (en) * 2014-11-21 2020-02-26 三菱ケミカル株式会社 Method for producing composite photocatalyst and composite photocatalyst
WO2023102096A1 (en) * 2021-12-03 2023-06-08 Merck Sharp & Dohme Llc Blocking and non-blocking single domain antibodies and uses thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6398384A (en) * 1986-10-13 1988-04-28 Unitika Ltd Immobilization of enzyme
JPH05276945A (en) * 1992-04-03 1993-10-26 Unitika Ltd Production of formed silicone material holding immobilized physiologically active substance
JPH07236690A (en) * 1994-02-25 1995-09-12 Unitika Ltd Method for fixing fibrinogenolysis active material
WO2002034301A1 (en) * 2000-10-20 2002-05-02 Noritake Co., Limited Material for treating harmful substance, and method and apparatus for treating harmful substance
WO2003033143A1 (en) * 2001-10-10 2003-04-24 Noritake Co.,Limited Photocatalytic material selectively inactivating biologically harmful substance and utilization thereof
WO2003033145A1 (en) * 2001-10-18 2003-04-24 Noritake Co.,Limited Photocatalytic material and utilization thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985656A (en) * 1986-06-12 1999-11-16 The Board Of Trustees Of The Leland Stanford Junior University Suppressor and progenitor cells
GB9007408D0 (en) * 1990-04-02 1990-05-30 Nycomed As Compositions
US5445970A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members
US6470220B1 (en) * 1999-03-29 2002-10-22 The Regents Of The University Of California Diagnosis and treatment of cancers using in vivo magnetic domains
US6203955B1 (en) * 2000-04-28 2001-03-20 Toshiba Tec Kabushiki Kaisha Developing agent and image forming apparatus
US6997863B2 (en) * 2001-07-25 2006-02-14 Triton Biosystems, Inc. Thermotherapy via targeted delivery of nanoscale magnetic particles

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6398384A (en) * 1986-10-13 1988-04-28 Unitika Ltd Immobilization of enzyme
JPH05276945A (en) * 1992-04-03 1993-10-26 Unitika Ltd Production of formed silicone material holding immobilized physiologically active substance
JPH07236690A (en) * 1994-02-25 1995-09-12 Unitika Ltd Method for fixing fibrinogenolysis active material
WO2002034301A1 (en) * 2000-10-20 2002-05-02 Noritake Co., Limited Material for treating harmful substance, and method and apparatus for treating harmful substance
WO2003033143A1 (en) * 2001-10-10 2003-04-24 Noritake Co.,Limited Photocatalytic material selectively inactivating biologically harmful substance and utilization thereof
WO2003033145A1 (en) * 2001-10-18 2003-04-24 Noritake Co.,Limited Photocatalytic material and utilization thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1614694A4 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005289660A (en) * 2004-03-31 2005-10-20 Toto Ltd Surface-modified titanium dioxide particulate and dispersion thereof and method for manufacturing the same
JP2008535788A (en) * 2005-02-28 2008-09-04 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Long-lasting water-resistant sunscreen comprising a metal oxide and a peptide conditioner
WO2007048635A1 (en) * 2005-10-28 2007-05-03 Abdula Kurkayev Method of activating a photosensitizer
WO2007048634A2 (en) 2005-10-28 2007-05-03 Abdula Kurkayev Nanoparticles of a heterocrystal mineral for use as a medicament and method of producing the same
WO2007048634A3 (en) * 2005-10-28 2007-12-06 Abdula Kurkayev Nanoparticles of a heterocrystal mineral for use as a medicament and method of producing the same
EP1779855A1 (en) * 2005-10-28 2007-05-02 Abdula Kurkayev Nanoparticles of a heterocrystal mineral for use as a medicament and method of producing the same
US7935329B2 (en) * 2005-12-20 2011-05-03 Lg Chem, Ltd. Titania sol, method of preparing the same, and coating composition comprising the same
WO2007122956A1 (en) * 2006-03-24 2007-11-01 Toto Ltd. Titanium oxide complex particle, dispersion solution of the particle, and process for production of the particle
WO2008111538A1 (en) * 2007-03-09 2008-09-18 Kazufumi Ogawa Magnetic-sensitive material, magnetic-sensitive medicinal agent, and those production methods
JP2008222595A (en) * 2007-03-09 2008-09-25 Kagawa Univ Magnetism-sensitive material, magnetism-sensitive medicament and methods for producing them
WO2009144775A1 (en) * 2008-05-29 2009-12-03 Toto株式会社 Anti-tumor agent
WO2010016581A1 (en) * 2008-08-08 2010-02-11 Toto株式会社 Ultrasonic cancer therapy accelerator
WO2011102407A1 (en) 2010-02-17 2011-08-25 国立大学法人神戸大学 Radiation therapy agent
US8580312B2 (en) 2010-02-17 2013-11-12 National University Corporation Kobe University Radiation therapy agent
JP2017122684A (en) * 2016-01-08 2017-07-13 積水メディカル株式会社 Immunoagglutination particles and method for manufacturing the particles

Also Published As

Publication number Publication date
JPWO2004087765A1 (en) 2006-10-05
AU2004226053A1 (en) 2004-10-14
EP1614694A1 (en) 2006-01-11
EP1614694A4 (en) 2008-11-05
US20060281087A1 (en) 2006-12-14

Similar Documents

Publication Publication Date Title
WO2004087765A1 (en) Titanium dioxide complex having molecule distinguishability
KR100796985B1 (en) Multi-Functional Nanoparticles Partially-Deposited with Gold Film
CN104688773A (en) Cell killer
EP2000150B1 (en) Titanium oxide complex particle, dispersion solution of the particle, and process for production of the particle
Voliani et al. Smart delivery and controlled drug release with gold nanoparticles: new frontiers in nanomedicine
US20080261805A1 (en) Photocatalytic Titanium Dioxide Microparticle, Dispersion Liquid Thereof and Process for Producing the Same
Ren et al. Synthesis of nir-sensitive Au–Au2S nanocolloids for drug delivery
JP3835700B2 (en) Dispersion containing titanium dioxide composite with molecular discrimination
JP7193461B2 (en) nanoparticles
JP2009091345A (en) Titanium oxide functional molecule composite particle
WO2010016581A1 (en) Ultrasonic cancer therapy accelerator
WO2009144775A1 (en) Anti-tumor agent
CN101346149A (en) Ultrasonic cancer treatment enhancer and cell killer
JP5044150B2 (en) Pharmaceutical titanium dioxide composite that loses its efficacy by light irradiation
CN100379761C (en) Titanium dioxide complex having molecule distinguishability
JP4169078B2 (en) Titanium oxide composite particles, dispersion thereof, and production method thereof
JP3826402B2 (en) Dispersion containing photocatalytic titanium dioxide composite fine particles
JP2008094816A (en) Ultrasonic cancer therapy promoter
CN114903844B (en) Hydrogel drug-carrying system, preparation method, application and pharmaceutical composition
Shringirishi et al. Gold nanoparticles: Promising and potential nanomaterial
JP4103929B2 (en) Cellicide
JP5224531B2 (en) Treatment for bladder cancer
Titova et al. APPLICATION OF GOLD NANOPARTICLES IN THE DIAGNOSIS OF ONCOLOGICAL DISEASES
Lu Advanced process and methods of drug delivery process
Carraro et al. MOFs and Biomacromolecules for Biomedical Applications

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 20048080532

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2005504274

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2004226053

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2004724768

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2004226053

Country of ref document: AU

Date of ref document: 20040331

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 2004226053

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2004724768

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2006281087

Country of ref document: US

Ref document number: 10551164

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 10551164

Country of ref document: US